Lipid-based nanoparticles and use of same in optimized insulin dosing regimens

ABSTRACT

The invention provides methods of treating a subject having diabetes mellitus and/or a metabolic derangement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 16/846,101, filed Apr. 10, 2020, nowallowed, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Applications No. 62/833,228, filed Apr. 12, 2019, andNo. 62/988,748, filed Mar. 12, 2020, all of which are herebyincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Phospholipid nanoparticles of diameter lower than about 100 nm are oftenused as carriers to improve in vivo delivery of active pharmaceuticalingredients (APIs), such as peptides and biogenic amines. Thenanoparticles' small particle size allows them to easily cross membranebarriers. Further, nanoparticles may provide rapid and specific deliveryof APIs to desired cell surface receptors, resulting in improvedpharmacological action and need for lower API doses. The targeted APIdelivery also leads to lower toxicity, because of the API's reduceddelivery to unwanted tissues in the body.

An example of such nanoparticles is the hepatic delivery vesicle (HDV),which comprises a hepatocyte-targeting component and delivers APIs tohepatocyte receptors. In contrast, nanoparticles without ahepatocyte-targeting components generally accumulate in livermacrophages called Kupffer cells, along with other macrophage cells inthe body.

Diabetes mellitus, encompassing Type 1 and Type 2 forms, is a disorderaffecting large numbers of people worldwide. Diabetes mellitusmanagement comprises normalizing blood glucose levels in the subject,and that may require multiple daily injections of an insulin-basedproduct. Despite the presence of various insulin-based products on themarket, there is still a need for novel insulin-containing formulationsthat control glucose blood levels in the subject over a wide period oftime.

Certain medications approved for insulin-requiring diabetes mellitustreatment comprise an insulin analog that is to be administeredsubcutaneously, often as a time-release formulation. Because of theabundance of insulin receptors in peripheral adipose and muscle tissues,such administration releases the insulin analog to peripheral tissues,but generally not to the liver. In one aspect, proper insulin-requiringdiabetes mellitus treatment requires an insulin-based formulation inwhich a portion of the dosed insulin is released to peripheral tissuesthroughout the day and another portion of the dosed insulin is targetedfor liver delivery. Such need extends as well to other therapeuticagents for which targeted liver delivery has advantageous therapeuticand/or pharmacological properties.

There is thus an unmet need in the art for compositions and methods foradministering insulin to a subject, such that the insulin is deliveredto peripheral tissues as well as to the liver of the subject. Suchcompositions and methods can be used to manage blood glucose levels inType 1 and Type 2 diabetic patients, as well as patients with metabolicderangements, such as but not limited to metabolic syndrome withelevated insulin levels, steatosis, and/or steatohepatitis. The presentinvention meets this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides in one aspect a method of optimizing the amountof bolus insulin and basal insulin to be administered to a subjecthaving diabetes mellitus, wherein the subject is administered an amountof a bolus insulin HDV composition comprising a lipid-basednanoparticle, wherein the bolus insulin is dispersed within thenanoparticle, wherein the subject is further administered an amount ofbasal insulin.

In certain embodiments, the method comprises varying the administeredamount of the bolus insulin HDV composition and the administered amountof the basal insulin so as to identify the optimized amount of the bolusinsulin HDV composition and the optimized amount of the basal insulin tobe administered to the subject to afford therapeutically effective bloodglucose control without significant hypoglycemia. In certainembodiments, the nanoparticle is enclosed by a bipolar lipid membranecomprising cholesterol, dicetyl phosphate, an amphipathic lipid, and ahepatocyte receptor binding molecule. In certain embodiments, theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In certain embodiments, theat least one hepatocyte receptor binding molecule extends outward fromthe nanoparticle. In certain embodiments, the size of the nanoparticleranges from about 10 nm to about 150 nm.

The invention provides in one aspect a method of optimizing the amountof bolus insulin and basal insulin to be administered to a subjecthaving diabetes, wherein the subject is originally administered anamount of bolus insulin and an amount of basal insulin such that thediabetes is well controlled in the subject.

In certain embodiments, the method comprises reducing the amount ofbasal insulin administered to the subject and varying the administeredamount of a bolus insulin HDV composition so as to identify theoptimized amount of the bolus insulin HDV composition and the optimizedamount of the basal insulin to be administered to the subject such thatthe diabetes is well controlled in the subject. In certain embodiments,the bolus insulin HDV composition comprises a lipid-based nanoparticle,wherein the bolus insulin is dispersed within the nanoparticle. Incertain embodiments, the nanoparticle is enclosed by a bipolar lipidmembrane comprising cholesterol, dicetyl phosphate, an amphipathiclipid, and a hepatocyte receptor binding molecule. In certainembodiments, the amphipathic lipid comprises at least one selected fromthe group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In certain embodiments, theat least one hepatocyte receptor binding molecule extends outward fromthe nanoparticle. In certain embodiments, the size of the nanoparticleranges from about 10 nm to about 150 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, there are depicted inthe drawings certain embodiments of the invention. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIGS. 1A-1F illustrate changes in hypoglycemia, A1c, and insulin bybaseline A1c. p-Values indicate significance of between-groupdifferences at endpoint.

FIG. 2 illustrates selected results of continuous glucose monitoring(CGM) studies in Example 1, in terms of % time that the patient hasblood glucose levels below 54 mg/dL vs. the patient's A1c level.

FIG. 3 is an illustrative scheme for a Phase II dose optimization studyin lower A1C patients (6.5-8.5% A1C).

FIG. 4 illustrates median insulin dosing results for Example 3.

FIG. 5 illustrates hypoglycemic events per week (defined as >15 Min CGM<54 mg/dL) for Example 3.

FIG. 6 illustrates hypoglycemic events per week (for day and night) forExample 3.

FIG. 7 illustrates change from baseline (Visit 5) in weight (kg) atVisit 11 for Example 3.

FIG. 8 illustrates change in mean glucose from optimized baseline(baseline=mean of Visits 4&5) for Example 3.

FIG. 9 illustrates bolus: basal insulin ratios for Example 3.

FIG. 10 illustrates results for Example 3 relating to hypoglycemic eventresults. In this study, a ninety-day unblinded CGM, followed by anoptimized standard of care, resulted in less hypoglycemia events and0.4% A1C reduction. When HDV was added to unblinded CGM, subjects inboth treatment groups achieved continued decreases in hypoglycemiaevents, despite using more insulin overall. Despite 10% or 40%reductions at Day 91 in basal insulin, both treatment groups' basaldosing returned to baseline levels by end of the study. Reductions inhypoglycemia during HDV treatment did not result in increased overallglycemia.

FIG. 11 illustrates results for Example 3 relating to hypoglycemic eventresults.

FIG. 12 illustrates results for Example 3 relating to reduction inhypoglycemic events in relation to baseline. Based on the studies usingunblinded CGM and optimized standard of care, a 0.4% A1C improvement wasobserved after 90 days, with an about 11% decrease in 24 hr and daytimehypoglycemic events, and with an about 20% decrease in nighttimehypoglycemic events. The addition of HDV to unblinded CGM allowed foradditional 17% decrease in 24 hr hypoglycemic events, additional 6%decrease in daytime hypoglycemic events, and additional 25% decrease innighttime hypoglycemic events. The addition of HDV therapy providedhypoglycemia benefit despite the facts that subjects used slightlyhigher overall insulin dosing and showed essentially no change in A1C.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in part to the unexpected discovery thatHDV-insulin enables hepatic metabolism of ingested carbohydrate(glucose), reducing the glucose load to peripheral tissues, thusrequiring an adjustment of basal doses of insulin so that fastinghypoglycemia is reduced or eliminated. The present invention provides,in one aspect, a new, physiologically adjusted ratio of meal-time bolusHDV-insulin dose to the 24-hour basal insulin, such as but not limitedto degludec.

In certain embodiments, the use of the HDV-insulin potentiates theeffect on insulin in the subject, allowing for use of lower amounts ofinsulin and thus avoiding iatrogenic hyperinsulinemia and/orhypoglycemia in the subject.

In certain embodiments, the use of the HDV-insulin allows for use oflower amounts of insulin, and thus reduce or eliminates side effectsassociated with hyperinsulinemia (which can be derived from use of largeamounts of insulin), such as but not limited to hypoglycemia, increasedrisk of polycystic ovary syndrome (PCOS), increased synthesis of VLDL(hypertriglyceridemia), hypertension (insulin increases sodium retentionby the renal tubules), coronary artery disease (increased insulindamages endothelial cells), increased risk of cardiovascular disease,and/or weight gain and lethargy.

In certain embodiments, the use of HDV-insulin allows for efficacious,yet intermittent transient, engagement of hepatic liver insulinreceptors for the improvement of hepatic metabolic function. In anon-limited, the HDV-insulin is administered to the patient around mealtime and allows for insulin to be delivered to the hepatic insulinreceptors during digestion. The HDV-insulin is eventually removed fromcirculation through natural metabolic processes and thus does notpromote constitutive engagement of hepatic liver insulin receptors, suchas PEG-lispro, which remains in circulation for prolonged periods oftime, much after the need to mealtime insulin has ceased.

Without wishing to be limited by any theory, the standard treatment of adiabetic subject involves a 50:50 (or 1:1) ratio of administered bolusinsulin and basal insulin. Using HDV in at least the bolus insulinallows for an insulin ratio that is closer to physiological levels (suchas, for example, using lower basal insulin amounts).

In certain embodiments, the present invention provides a method ofoptimizing the amount of bolus insulin and basal insulin to beadministered to a subject having diabetes mellitus. In otherembodiments, the subject is (initially) administered an amount of abolus insulin HDV composition comprising a lipid-based nanoparticle,wherein the bolus insulin is dispersed within the nanoparticle. In yetother embodiments, the subject is (initially) further administered anamount of basal insulin. In yet other embodiments, the method of theinvention comprises varying the administered amount of the bolus insulinHDV composition and the administered amount of the basal insulin so asto identify the amount of the bolus insulin HDV composition and theamount of the basal insulin to be administered to the subject to affordtherapeutically effective blood glucose control without significanthypoglycemia.

In certain embodiments, the present invention provides a method ofoptimizing the amount of bolus insulin and basal insulin to beadministered to a subject having diabetes, wherein the subject isoriginally administered an amount of bolus insulin and an amount ofbasal insulin such that the diabetes is well controlled in the subject.In other embodiments, the method comprises reducing the amount of basalinsulin administered to the subject and varying the administered amountof a bolus insulin HDV composition so as to identify the optimizedamount of the bolus insulin HDV composition and the optimized amount ofthe basal insulin to be administered to the subject such that thediabetes is well controlled in the subject. In yet other embodiments,the bolus insulin HDV composition comprises a lipid-based nanoparticle,wherein the bolus insulin is dispersed within the nanoparticle. In yetother embodiments, the nanoparticle is enclosed by a bipolar lipidmembrane comprising cholesterol, dicetyl phosphate, an amphipathiclipid, and a hepatocyte receptor binding molecule. In yet otherembodiments, the amphipathic lipid comprises at least one selected fromthe group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In yet other embodiments,the at least one hepatocyte receptor binding molecule extends outwardfrom the nanoparticle. In yet other embodiments, the size of thenanoparticle ranges from about 10 nm to about 150 nm.

In certain embodiments, the diabetes is diabetes mellitus.

In certain embodiments, the subject has about 6.5-8.5% A1C. In certainembodiments, the subject has 70-120 mg/dL fasting blood sugar. Incertain embodiments, the subject has 80-110 mg/dL fasting blood sugar.In certain embodiments, the subject has 80-100 mg/dL fasting bloodsugar. In certain embodiments, the subject experiences fewerhypoglycemia as compared to the treatment without HDV. In certainembodiments, the reduction in the amount of bolus insulin is about 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or 80%. In certain embodiments, the reduction in the amount ofbolus insulin ranges from about 10% to about 40%. In certainembodiments, the subject experiences weight loss as compared to thetreatment without HDV.

In certain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition (i.e., the amount of bolusinsulin in the HDV composition) and the administered basal insulindepends in the severity of diabetes mellitus, which can be measured in anon-limiting embodiment by hemoglobin A1c (HbA1c). In certainembodiments, the optimized insulin ratio between the administered bolusinsulin HDV composition and the administered basal insulin is equal to,or greater than, about 1:1 when the subject has >8.5% HbA1c. In certainembodiments, the optimized insulin ratio between the administered bolusinsulin HDV composition and the administered basal insulin is equal to,or lower than, about 1:1 when the subject has <8.5% HbA1c. In certainembodiments, the optimized insulin ratio between the administered bolusinsulin HDV composition and the administered basal insulin is equal to,or greater than, about 1:1 when the subject has <8.5% HbA1c. In certainembodiments, the optimized insulin ratio between the administered bolusinsulin HDV composition and the administered basal insulin is equal to,or lower than, about 1:1 when the subject has >8.5% HbA1c.

In certain embodiments, the subject has a HbA1c level equal to orgreater than about 10%. In certain embodiments, the subject has a HbA1clevel equal to or greater than about 9.9%. In certain embodiments, thesubject has a HbA1c level equal to or greater than about 9.8%. Incertain embodiments, the subject has a HbA1c level equal to or greaterthan about 9.7%. In certain embodiments, the subject has a HbA1c levelequal to or greater than about 9.6%. In certain embodiments, the subjecthas a HbA1c level equal to or greater than about 9.5%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 9.4%. In certain embodiments, the subject has a HbA1c level equalto or greater than about 9.3%. In certain embodiments, the subject has aHbA1c level equal to or greater than about 9.2%. In certain embodiments,the subject has a HbA1c level equal to or greater than about 9.1%. Incertain embodiments, the subject has a HbA1c level equal to or greaterthan about 9.0%. In certain embodiments, the subject has a HbA1c levelequal to or greater than about 8.9%. In certain embodiments, the subjecthas a HbA1c level equal to or greater than about 8.8%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 8.7%. In certain embodiments, the subject has a HbA1c level equalto or greater than about 8.6%. In certain embodiments, the subject has aHbA1c level equal to or greater than about 8.5%. In certain embodiments,the subject has a HbA1c level equal to or greater than about 8.4%. Incertain embodiments, the subject has a HbA1c level equal to or greaterthan about 8.3%. In certain embodiments, the subject has a HbA1c levelequal to or greater than about 8.2%. In certain embodiments, the subjecthas a HbA1c level equal to or greater than about 8.1%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 8.0%. In certain embodiments, the subject has a HbA1c level equalto or greater than about 7.9%. In certain embodiments, the subject has aHbA1c level equal to or greater than about 7.8%. In certain embodiments,the subject has a HbA1c level equal to or greater than about 7.7%. Incertain embodiments, the subject has a HbA1c level equal to or greaterthan about 7.6%. In certain embodiments, the subject has a HbA1c levelequal to or greater than about 7.5%. In certain embodiments, the subjecthas a HbA1c level equal to or greater than about 7.4%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 7.3%. In certain embodiments, the subject has a HbA1c level equalto or greater than about 7.2%. In certain embodiments, the subject has aHbA1c level equal to or greater than about 7.1%. In certain embodiments,the subject has a HbA1c level equal to or greater than about 7.0%. Incertain embodiments, the subject has a HbA1c level equal to or greaterthan about 6.9%. In certain embodiments, the subject has a HbA1c levelequal to or greater than about 6.8%. In certain embodiments, the subjecthas a HbA1c level equal to or greater than about 6.7%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 6.6%. In certain embodiments, the subject has a HbA1c level equalto or greater than about 6.5%.

In certain embodiments, the subject has a HbA1c level equal to or lowerthan about 10%. In certain embodiments, the subject has a HbA1c levelequal to or lower than about 9.9%. In certain embodiments, the subjecthas a HbA1c level equal to or lower than about 9.8%. In certainembodiments, the subject has a HbA1c level equal to or lower than about9.7%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 9.6%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 9.5%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 9.4%. In certainembodiments, the subject has a HbA1c level equal to or lower than about9.3%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 9.2%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 9.1%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 9.0%. In certainembodiments, the subject has a HbA1c level equal to or lower than about8.9%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 8.8%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 8.7%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 8.6%. In certainembodiments, the subject has a HbA1c level equal to or lower than about8.5%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 8.4%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 8.3%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 8.2%. In certainembodiments, the subject has a HbA1c level equal to or lower than about8.1%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 8.0%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 7.9%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 7.8%. In certainembodiments, the subject has a HbA1c level equal to or lower than about7.7%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 7.6%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 7.5%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 7.4%. In certainembodiments, the subject has a HbA1c level equal to or lower than about7.3%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 7.2%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 7.1%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 7.0%. In certainembodiments, the subject has a HbA1c level equal to or lower than about6.9%. In certain embodiments, the subject has a HbA1c level equal to orlower than about 6.8%. In certain embodiments, the subject has a HbA1clevel equal to or lower than about 6.7%. In certain embodiments, thesubject has a HbA1c level equal to or lower than about 6.6%. In certainembodiments, the subject has a HbA1c level equal to or greater thanabout 6.5%.

In certain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.1. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.15. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.2. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.25. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.3. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.35. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.4. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.45. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.5. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.55. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.6. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.65. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.7. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.75. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.8. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:0.85. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:0.9. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:0.95. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.05. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.1. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.15. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.2. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.25. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.3. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.35. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.4. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.45. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.5. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.55. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.6. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.65. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.7. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.75. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.8. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:1.85. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:1.9. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:1.95. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:2. In certainembodiments, the optimized insulin ratio between the administered bolusinsulin HDV composition and the administered basal insulin is about1:2.05. In certain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:2.1. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:2.2. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:2.3. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:2.4. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:2.5. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:2.6. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 2.7. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:2.8. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:2.9. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:3. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:3.1. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:3.2. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:3.3. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:3.4. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:3.5. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:3.6. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:3.7. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:3.8. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:3.9. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:4.0. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:4.1. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:4.2. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:4.3. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:4.4. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:4.5. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:4.6. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:4.7. Incertain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is about 1:4.8. In certain embodiments, the optimized insulinratio between the administered bolus insulin HDV composition and theadministered basal insulin is about 1:4.9. In certain embodiments, theoptimized insulin ratio between the administered bolus insulin HDVcomposition and the administered basal insulin is about 1:5.

In certain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is equal to, or greater than, about 1:0.1, about 1:0.15, about1:0.2, about 1:0.25, about 1:0.3, about 1:0.35, about 1:0.4, about1:0.45, about 1:0.5, about 1:0.55, about 1:0.6, about 1:0.65, about1:0.7, about 1:0.75, about 1:0.8, about 1:0.85, about 1:0.9, about1:0.95, about 1:1, about 1:1.05, about 1:1.1, about 1:1.15, about 1:1.2,about 1:1.25, about 1:1.3, about 1:1.35, about 1:1.4, about 1:1.45,about 1:1.5, about 1:1.55, about 1:1.6, about 1:1.65, about 1:1.7, about1:1.75, about 1:1.8, about 1:1.85, about 1:1.9, about 1:1.95, about 1:2,about 1:2.05, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about1:2.5, about 1:2.6, about 2.7, about 1:2.8, about 1:2.9, about 1:3,about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4, about 1:3.5, about1:3.6, about 1:3.7, about 1:3.8, about 1:3.9, about 1:4.0, about 1:4.1,about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about1:4.7, about 1:4.8, about 1:4.9, and/or about 1:5.

In certain embodiments, the optimized insulin ratio between theadministered bolus insulin HDV composition and the administered basalinsulin is equal to, or lower than, about 1:0.1, about 1:0.15, about1:0.2, about 1:0.25, about 1:0.3, about 1:0.35, about 1:0.4, about1:0.45, about 1:0.5, about 1:0.55, about 1:0.6, about 1:0.65, about1:0.7, about 1:0.75, about 1:0.8, about 1:0.85, about 1:0.9, about1:0.95, about 1:1, about 1:1.05, about 1:1.1, about 1:1.15, about 1:1.2,about 1:1.25, about 1:1.3, about 1:1.35, about 1:1.4, about 1:1.45,about 1:1.5, about 1:1.55, about 1:1.6, about 1:1.65, about 1:1.7, about1:1.75, about 1:1.8, about 1:1.85, about 1:1.9, about 1:1.95, about 1:2,about 1:2.05, about 1:2.1, about 1:2.2, about 1:2.3, about 1:2.4, about1:2.5, about 1:2.6, about 2.7, about 1:2.8, about 1:2.9, about 1:3,about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4, about 1:3.5, about1:3.6, about 1:3.7, about 1:3.8, about 1:3.9, about 1:4.0, about 1:4.1,about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about1:4.7, about 1:4.8, about 1:4.9, and/or about 1:5.

In certain embodiments, the dose of insulin is per day (daily).

In certain embodiments, the dose of insulin is about 0.01 units/kg. Incertain embodiments, the dose of insulin is about 0.02 units/kg. Incertain embodiments, the dose of insulin is about 0.03 units/kg. Incertain embodiments, the dose of insulin is about 0.04 units/kg. Incertain embodiments, the dose of insulin is about 0.05 units/kg. Incertain embodiments, the dose of insulin is about 0.06 units/kg. Incertain embodiments, the dose of insulin is about 0.07 units/kg. Incertain embodiments, the dose of insulin is about 0.08 units/kg. Incertain embodiments, the dose of insulin is about 0.09 units/kg. Incertain embodiments, the dose of insulin is about 0.1 units/kg. Incertain embodiments, the dose of insulin is about 0.15 units/kg. Incertain embodiments, the dose of insulin is about 0.2 units/kg. Incertain embodiments, the dose of insulin is about 0.25 units/kg. Incertain embodiments, the dose of insulin is about 0.3 units/kg. Incertain embodiments, the dose of insulin is about 0.35 units/kg. Incertain embodiments, the dose of insulin is about 0.4 units/kg. Incertain embodiments, the dose of insulin is about 0.45 units/kg. Incertain embodiments, the dose of insulin is about 0.5 units/kg. Incertain embodiments, the dose of insulin is about 0.55 units/kg. Incertain embodiments, the dose of insulin is about 0.6 units/kg. Incertain embodiments, the dose of insulin is about 0.65 units/kg. Incertain embodiments, the dose of insulin is about 0.7 units/kg. Incertain embodiments, the dose of insulin is about 0.75 units/kg. Incertain embodiments, the dose of insulin is about 0.8 units/kg. Incertain embodiments, the dose of insulin is about 0.85 units/kg. Incertain embodiments, the dose of insulin is about 0.9 units/kg. Incertain embodiments, the dose of insulin is about 0.95 units/kg. Incertain embodiments, the dose of insulin is about 1 unit/kg. In certainembodiments, the dose of insulin is about 1.1 units/kg. In certainembodiments, the dose of insulin is about 1.2 units/kg. In certainembodiments, the dose of insulin is about 1.3 units/kg. In certainembodiments, the dose of insulin is about 1.4 units/kg. In certainembodiments, the dose of insulin is about 1.5 units/kg. In certainembodiments, the dose of insulin is about 1.6 units/kg. In certainembodiments, the dose of insulin is about 1.7 units/kg. In certainembodiments, the dose of insulin is about 1.8 units/kg. In certainembodiments, the dose of insulin is about 1.9 units/kg. In certainembodiments, the dose of insulin is about 2 units/kg. In certainembodiments, the dose of insulin is about 2.1 units/kg. In certainembodiments, the dose of insulin is about 2.2 units/kg. In certainembodiments, the dose of insulin is about 2.3 units/kg. In certainembodiments, the dose of insulin is about 2.4 units/kg. In certainembodiments, the dose of insulin is about 2.5 units/kg. In certainembodiments, the dose of insulin is about 2.6 units/kg. In certainembodiments, the dose of insulin is about 2.7 units/kg. In certainembodiments, the dose of insulin is about 2.8 units/kg. In certainembodiments, the dose of insulin is about 2.9 units/kg. In certainembodiments, the dose of insulin is about 3.0 units/kg. In certainembodiments, the dose of insulin is about 3.2 units/kg. In certainembodiments, the dose of insulin is about 3.4 units/kg. In certainembodiments, the dose of insulin is about 3.5 units/kg. In certainembodiments, the dose of insulin is about 3.6 units/kg. In certainembodiments, the dose of insulin is about 3.8 units/kg. In certainembodiments, the dose of insulin is about 4 units/kg. In certainembodiments, the dose of insulin is about 4.5 units/kg. In certainembodiments, the dose of insulin is about 5 units/kg. In certainembodiments, the dose of insulin is about 5.5 units/kg. In certainembodiments, the dose of insulin is about 6 units/kg. In certainembodiments, the dose of insulin is about 6.5 units/kg. In certainembodiments, the dose of insulin is about 7 units/kg. In certainembodiments, the dose of insulin is about 7.5 units/kg. In certainembodiments, the dose of insulin is about 8 units/kg. In certainembodiments, the dose of insulin is about 8.5 units/kg. In certainembodiments, the dose of insulin is about 9 units/kg. In certainembodiments, the dose of insulin is about 9.5 units/kg. In certainembodiments, the dose of insulin is about 10 units/kg. In certainembodiments, the dose of insulin is about 11 units/kg. In certainembodiments, the dose of insulin is about 12 units/kg. In certainembodiments, the dose of insulin is about 13 units/kg. In certainembodiments, the dose of insulin is about 14 units/kg. In certainembodiments, the dose of insulin is about 15 units/kg. In certainembodiments, the dose of insulin is about 16 units/kg. In certainembodiments, the dose of insulin is about 17 units/kg. In certainembodiments, the dose of insulin is about 18 units/kg. In certainembodiments, the dose of insulin is about 19 units/kg. In certainembodiments, the dose of insulin is about 20 units/kg.

In certain embodiments, the dose of insulin is greater than about 0.01units/kg, about 0.02 units/kg, about 0.03 units/kg, about 0.04 units/kg,about 0.05 units/kg, about 0.06 units/kg, about 0.07 units/kg, about0.08 units/kg, about 0.09 units/kg, about 0.1 units/kg, about 0.15units/kg, about 0.2 units/kg, about 0.25 units/kg, about 0.3 units/kg,about 0.35 units/kg, about 0.4 units/kg, about 0.45 units/kg, about 0.5units/kg, about 0.55 units/kg, about 0.6 units/kg, about 0.65 units/kg,about 0.7 units/kg, about 0.75 units/kg, about 0.8 units/kg, about 0.85units/kg, about 0.9 units/kg, about 0.95 units/kg, about 1 unit/kg,about 1.1 units/kg, about 1.2 units/kg, about 1.3 units/kg, about 1.4units/kg, about 1.5 units/kg, about 1.6 units/kg, about 1.7 units/kg,about 1.8 units/kg, about 1.9 units/kg, about 2 units/kg, about 2.1units/kg, about 2.2 units/kg, about 2.3 units/kg, about 2.4 units/kg,about 2.5 units/kg, about 2.6 units/kg, about 2.7 units/kg, about 2.8units/kg, about 2.9 units/kg, about 3.0 units/kg, about 3.2 units/kg,about 3.4 units/kg, about 3.5 units/kg, about 3.6 units/kg, about 3.8units/kg, about 4 units/kg, about 4.5 units/kg, about 5 units/kg, about5.5 units/kg, about 6 units/kg, about 6.5 units/kg, about 7 units/kg,about 7.5 units/kg, about 8 units/kg, about 8.5 units/kg, about 9units/kg, about 9.5 units/kg, about 10 units/kg, about 11 units/kg,about 12 units/kg, about 13 units/kg, about 14 units/kg, about 15units/kg, about 16 units/kg, about 17 units/kg, about 18 units/kg, about19 units/kg, or about 20 units/kg.

In certain embodiments, the dose of insulin is lower than about 0.01units/kg, about 0.02 units/kg, about 0.03 units/kg, about 0.04 units/kg,about 0.05 units/kg, about 0.06 units/kg, about 0.07 units/kg, about0.08 units/kg, about 0.09 units/kg, about 0.1 units/kg, about 0.15units/kg, about 0.2 units/kg, about 0.25 units/kg, about 0.3 units/kg,about 0.35 units/kg, about 0.4 units/kg, about 0.45 units/kg, about 0.5units/kg, about 0.55 units/kg, about 0.6 units/kg, about 0.65 units/kg,about 0.7 units/kg, about 0.75 units/kg, about 0.8 units/kg, about 0.85units/kg, about 0.9 units/kg, about 0.95 units/kg, about 1 unit/kg,about 1.1 units/kg, about 1.2 units/kg, about 1.3 units/kg, about 1.4units/kg, about 1.5 units/kg, about 1.6 units/kg, about 1.7 units/kg,about 1.8 units/kg, about 1.9 units/kg, about 2 units/kg, about 2.1units/kg, about 2.2 units/kg, about 2.3 units/kg, about 2.4 units/kg,about 2.5 units/kg, about 2.6 units/kg, about 2.7 units/kg, about 2.8units/kg, about 2.9 units/kg, about 3.0 units/kg, about 3.2 units/kg,about 3.4 units/kg, about 3.5 units/kg, about 3.6 units/kg, about 3.8units/kg, about 4 units/kg, about 4.5 units/kg, about 5 units/kg, about5.5 units/kg, about 6 units/kg, about 6.5 units/kg, about 7 units/kg,about 7.5 units/kg, about 8 units/kg, about 8.5 units/kg, about 9units/kg, about 9.5 units/kg, about 10 units/kg, about 11 units/kg,about 12 units/kg, about 13 units/kg, about 14 units/kg, about 15units/kg, about 16 units/kg, about 17 units/kg, about 18 units/kg, about19 units/kg, or about 20 units/kg.

In certain embodiments, the nanoparticles useful within the inventionare described in U.S. Patent Application Nos. US20110135725 andUS20090087479 and PCT Patent Application Publication No. WO 2018/169954,all of which are incorporated herein in their entireties by reference.In certain embodiments, the reduced or minimal aggregation properties ofthe nanoparticle of the invention improves its stability andpharmaceutical developability as compared to nanoparticles of the priorart.

In certain embodiments, the lipid-based nanoparticle of the invention isdefined and/or enclosed by a bipolar lipid membrane. In otherembodiments, the nanoparticle of the invention comprises ahepatocyte-targeting compound, which helps deliver the therapeutic agent(such as, but not limited to, insulin) associated with, and/or dispersedwithin, the nanoparticle to a hepatocyte. In yet other embodiments, thenanoparticle of the invention is part of a composition furthercomprising a “free” therapeutic agent, which is not associated with,and/or dispersed within, the nanoparticle. The nanoparticle, and anycompositions comprising the same, can be administered by any compatibleand/or feasible routes, such as but not limited to by injection (suchas, for example, subcutaneously and/or transdermally), inhalationally,buccally and/or orally, so as to treat a subject that benefits fromadministration of the therapeutic agent associated with, and/ordispersed within, the nanoparticle, and/or of the “free” therapeuticagent, which is not associated with, and/or dispersed within, thenanoparticle.

In certain embodiments, the therapeutic agent comprises serotonin, or5-hydroxytryptamine (5-HT), which is a monoamine neurotransmitter.

In certain embodiments, the therapeutic agent comprises a glucagon-likepeptide-1 (GLP-1) agonist. GLP-1 is a potent incretin hormone producedin the L-cells of the distal ileum and colon. In the L-cells, GLP-1 isgenerated by tissue-specific posttranslational processing of theproglucagon gene. Nutrients, including glucose, fatty acids, and dietaryfiber, are all known to upregulate the transcription of the geneencoding GLP-1, and they can stimulate the release of this hormone. Thelevels of GLP-1 rise rapidly upon food ingestion. Nutrients, principallysugars and fats, liberate GLP-1 and GLP-1-releasing factors, includingglucose-dependent insulinotropic peptide (GIP), gastrin-releasingpeptide, and selective neural regulators that also stimulate GLP-1secretion. Non-limiting examples of GLP-1 agonists of interest areliraglutide, semaglutide, and repaglinide.

Liposomes usually comprise amphipathic phospholipid materials that formbilayer membranes that define and/or enclose the liposomes. They canhave a single membrane (unilamellar), or multiple bilayers with amicroscopic onion-like appearance. Liposomes can be rather large,measuring several microns in diameter. Liposomes generally have aspherical (or nearly spherical) shape, wherein the intact surface has noavailable “open” edges and thus cannot interact with other available“open” edge liposome(s) to undergo particle aggregation.

In contrast, phospholipid nanoparticles with diameters equal to or lowerthan about 200 nm have a restricted ability to bend into a sphericalconfiguration, which should in principle be their thermodynamicallystable structure. As a result, these low-diameter nanoparticles do notform a perfectly spherical particle, but rather a nearly planar sheet.Without wishing to be limited by any theory, those nearly planar sheetscan be described as “nanodiscs” or “nanodisks” or “nanoFrisbees” or“bicelles.” Such nanoparticles have “open” edges in their membranes, andthese “edges” promote nanoparticle aggregation. As a result, in manyinstances the nanoparticles are generated as discrete particles, whichthan proceed to aggregate into larger, easily visible (wispy orfeather-like) floating particles. This phenomenon may hamper thedevelopability of the low-diameter nanoparticles as drug deliveryagents. In certain embodiments, unlike in the case of liposomes, the APIis not carried in the core volume of (or within) the bicelles. In otherembodiments, the API is attached and/or bound to the membrane surface ofthe bicelles, either through a purely physical interaction or a covalentlinkage. In one aspect, the present invention addresses this issue,providing compositions and methods that allow for closing the “open”edges of the nearly planar sheets (nanodiscs and/or nanoFrisbees) andthus minimizing or suppressing their tendency to self-aggregate.

As described herein, in certain embodiments, the lipid-basednanoparticles of the invention are useful as pharmaceutical carriers,and do not form the wispy, feathery-like structures described elsewhereherein. In certain embodiments, the nanoparticles of the inventioncomprise certain amphipathic lipids and/or certain organic moleculesthat enable the “open” edges of the planar nanoparticle membranes to bechanged in a way that prevents aggregation of the nanoparticles.

In certain embodiments, appropriate closing of the “open” edges of thelipid-based nanoparticle is promoted by replacing a portion ofdistearoyl phosphatidylcholine [also known as(S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two C₁₈ acyl groups covalently linked to aglycerol backbone] with a C₁₂-C₂₄ acyl lysophosphatidylcholine [alsoknown as C₁₂-C₂₄ acyl lysolecithin, or 1-(C₁₂-C₂₄acyl)-sn-glycero-3-phosphocholine, or (S)-2-hydroxy-3-(C₁₂-C₂₄acyloxy)propyl (2-(trimethylammonio)ethyl) phosphate, which comprises asingle C₁₂-C₂₄ acyl group covalently linked to a glycerol backbone]:

In certain embodiments, appropriate closing of the “open” edges of thelipid-based nanoparticle is promoted by replacing a portion ofdistearoyl phosphatidylcholine [also known as(S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two C₁₈ acyl groups covalently linked to aglycerol backbone] with stearoyl lysophosphatidylcholine [also known as1-steroyl-sn-glycero-3-phosphocholine, or(S)-2-hydroxy-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate, which comprises a single C₁₈ acyl group covalently linked toa glycerol backbone]:

In certain embodiments, when incorporated into the membrane, a C₁₂-C₂₄acyl lysophosphatidylcholine (such as but not limited to stearoyllysophosphatidylcholine) prevents and/or minimizes the aggregation thatoccurs when that compound is omitted from the membrane. In otherembodiments, the C₁₂-C₂₄ acyl lysophosphatidylcholine (such as but notlimited to stearoyl lysophosphatidylcholine), with its single aliphaticchain, enables closure of any existing membrane “edge” in thenanoparticle.

In certain embodiments, when incorporated into the membrane, any ofcertain small molecule stabilizers or any salts and/or solvates thereof,such as but not limited to m-cresol, benzyl alcohol, methyl4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (also knownas 2,6-di-tert-butyl-4-methylphenol), prevents and/or minimizes theaggregation that occurs when that compound is omitted from the membrane.In other embodiments, the small molecule stabilizers or any salts and/orsolvates thereof enable closure of any existing membrane “edges” in thenanoparticle.

In certain embodiments, when incorporated into the membrane, anycombinations of any of certain small molecule stabilizers or any saltsand/or solvates thereof, and the C₁₂-C₂₄ acyl lysophosphatidylcholine,prevents and/or minimizes the aggregation that occurs when that compoundis omitted from the membrane.

Compositions

The invention provides lipid-based nanoparticles, and compositionscomprising the same. In certain embodiments, the nanoparticle comprises,and/or is defined by, a bipolar lipid membrane.

In certain embodiments, the membrane comprises cholesterol. In otherembodiments, the membrane comprises dicetyl phosphate. In yet otherembodiments, the membrane comprises an amphipathic lipid. In yet otherembodiments, the membrane comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In yet otherembodiments, the membrane comprises cholesterol, dicetyl phosphate, andDSPC. In yet other embodiments, the membrane comprises a hepatocytereceptor binding molecule.

In certain embodiments, the amphipathic lipid comprises at least oneselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In other embodiments, theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

In certain embodiments, the hepatocyte receptor binding moleculecomprises biotin. In other embodiments, the biotin-containing hepatocytereceptor binding molecule comprises at least one selected from the groupconsisting of N-hydroxysuccinimide (NHS) biotin; sulfo-NHS-biotin;N-hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide longchain biotin; D-biotin; biocytin; sulfo-N-hydroxysuccinimide-S—S-biotin;biotin-BMCC; biotin-HPDP; iodoacetyl-LC-biotin; biotin-hydrazide;biotin-LC-hydrazide; biocytin hydrazide; biotin cadaverine;carboxybiotin; photobiotin; p-aminobenzoyl biocytin trifluoroacetate;p-diazobenzoyl biocytin; biotin DHPE (2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.

In certain embodiments, the hepatocyte receptor binding molecule isselected form the group consisting of 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate (biotin DHPE) and biotin-X-DHPE (2,3-diacetoxy propyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate).

In certain embodiments, the cholesterol ranges from about 5% to about25% (w/w) in the membrane. In other embodiments, the cholesterol ispresent in the membrane at a concentration of about 5%, 5.5%, 6%, 6.5%,7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%,13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%,19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or25% (w/w).

In certain embodiments, the dicetyl phosphate ranges from about 10% toabout 25% (w/w) in the membrane. In other embodiments, the dicetylphosphate is present in the membrane at a concentration of about 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%,16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%,22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w/w).

In certain embodiments, the DSPC ranges from about 40% to about 75%(w/w) in the membrane. In other embodiments, the DSPC is present in themembrane at a concentration of about 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or75% (w/w).

In certain embodiments, the hepatocyte receptor binding molecule rangesfrom about 0.5% to about 10% (w/w) in the membrane. In otherembodiments, the hepatocyte receptor binding molecule is present in themembrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%,1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%,2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%,3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%,8%, 8.5%, 9%, 9.5%, or 10% (w/w).

In certain embodiments, the membrane comprises at least one compoundselected from the group consisting of a stabilizer and a C₁₂-C₂₄ acyllysophosphatidylcholine.

In certain embodiments, the membrane further comprises a C₁₂-C₂₄ acyllysophosphatidylcholine. In other embodiments, the membrane furthercomprises stearoyl lysophosphatidylcholine.

In certain embodiments, the membrane further comprises m-cresol.

In certain embodiments, the stabilizer is selected from the groupconsisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate,thiomersal, and butylated hydroxytoluene(2,6-di-tert-butyl-4-methylphenol).

In certain embodiments, the stabilizer ranges from about 10% to about25% (w/w) in the membrane. In other embodiments, the stabilizer ispresent in the membrane at a concentration of about 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the m-cresol ranges from about 10% to about 25%(w/w) in the membrane. In other embodiments, the m-cresol is present inthe membrane at a concentration of about 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the C₁₂-C₂₄ lysophosphatidylcholine ranges fromabout 5% to about 30% (w/w) in the membrane. In other embodiments, theC₁₂-C₂₄ lysophosphatidylcholine ranges from about 1% to about 30% (w/w)in the membrane. In yet other embodiments, the C₁₂-C₂₄lysophosphatidylcholine is present in the membrane at a concentration ofabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or30% (w/w).

In certain embodiments, the stearoyl lysophosphatidylcholine ranges fromabout 5% to about 30% (w/w) in the membrane. In other embodiments, thestearoyl lysophosphatidylcholine ranges from about 1% to about 30% (w/w)in the membrane. In yet other embodiments, the stearoyllysophosphatidylcholine is present in the membrane at a concentration ofabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or30% (w/w).

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1% to about 30% (w/w)of the amount of DSPC in the membrane. In yet other embodiments, theamount of the C₁₂-C₂₄ lysophosphatidylcholine in the membrane is about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29% (w/w) or 30% (w/w) of the amount of DSPC in the membrane.

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of DSPC in the membrane. In yet other embodiments,the amount of the C₁₂-C₂₄ lysophosphatidylcholine in the membrane isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50mole % of the amount of DSPC in the membrane.

In certain embodiments, the amount of the stearoyllysophosphatidylcholine in the membrane is about 1% to about 30% (w/w)of the amount of DSPC in the membrane. In yet other embodiments, theamount of the stearoyl lysophosphatidylcholine in the membrane is about1%, 6%, 7%, 8%, 9%, 10%−11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w/w) of theamount of DSPC in the membrane.

In certain embodiments, the amount of the stearoyllysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of DSPC in the membrane. In yet other embodiments,the amount of the stearoyl lysophosphatidylcholine in the membrane isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50mole % of the amount of DSPC in the membrane.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE. In other embodiments, the membrane comprises cholesterol,dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, andbiotin DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, m-cresol, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE. In other embodiments, themembrane comprises cholesterol, dicetyl phosphate, DSPC, m-cresol, andbiotin DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and at least oneselected from the group consisting of biotin DHPE and biotin-X-DHPE. Inother embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE.

In certain embodiments, the stabilizer is contacted with the membrane,and/or the lipid components that assemble to form the membrane (such as,but not limited to, cholesterol, dicetyl phosphate, DSPC, C₁₂-C₂₄lysophosphatidylcholine if present, and biotin DHPE), at a (w/w) ratioof the membrane to the stabilizer ranging from about 1:1 to about 1:30.In other embodiments, the stabilizer is contacted with the membrane,and/or the lipid components that assemble to form the membrane, at a(w/w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2,1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8,1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or1:30.

In certain embodiments, the m-cresol is contacted with the membrane,and/or the lipid components that assemble to form the membrane (such as,but not limited to, cholesterol, dicetyl phosphate, DSPC, C₁₂-C₂₄lysophosphatidylcholine if present, and biotin DHPE), at a (w/w) ratioof the membrane to the stabilizer ranging from about 1:1 to about 1:30.In other embodiments, the m-cresol is contacted with the membrane,and/or the lipid components that assemble to form the membrane, at a(w/w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2,1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8,1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or1:30.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 9.4:18.1:56.8:14.1:0.0:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE, in a% (w/w) ratio of about 9.4:18.1:56.8:14.1:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 7.7:15.0:58.6:0.0:17.4:1.3.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, and biotin DHPE, in a % (w/w) ratio of about9.3:18.2:71.0:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 8.4:16.2:47.5:7.6:19.0:1.3.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE, in a% (w/w) ratio of about 10.4:20:58.6:9.4:1.6.

In certain embodiments, the at least one hepatocyte receptor bindingmolecule extends outward from the nanoparticle.

The invention should not be construed to be limited to the constructsdescribed and/or exemplified herein. Rather, the invention providesmethods of stabilizing and/or preventing aggregation of liposomes andother lipid-based nanoparticles, wherein the membrane is contacted withat least one selected from the group consisting of a stabilizer and aC₁₂-C₂₄ acyl lysophosphatidylcholine. In certain embodiments, thecontacting removes or minimizes any “free” edges in the membrane thatlead to aggregation of the liposomes and other lipid-basednanoparticles.

In certain embodiments, the stabilizer is selected from the groupconsisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate,thiomersal, and butylated hydroxytoluene. In other embodiments, thestabilizer, such as but not limited to m-cresol, ranges from about 10%to about 25% (w/w) in the membrane. In yet other embodiments, thestabilizer, such as but not limited to m-cresol, is present in themembrane at a concentration of about 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the C₁₂-C₂₄ lysophosphatidylcholine, such as butnot limited to stearoyl lysophosphatidylcholine, ranges from about 5% toabout 30% (w/w) in the membrane. In other embodiments, the C₁₂-C₂₄lysophosphatidylcholine, such as but not limited to stearoyllysophosphatidylcholine, ranges from about 1% to about 30% (w/w) in themembrane. In yet other embodiments, the C₁₂-C₂₄ lysophosphatidylcholine,such as but not limited to stearoyl lysophosphatidylcholine, is presentin the membrane at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w/w).

In certain embodiments, the membrane comprises at least one amphipathiclipid selected to from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In other embodiments, theamphipathic lipid is at least one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1%-30% (w/w) of theamount of the at least one amphipathic lipid in the membrane. In yetother embodiments, the amount of the C₁₂-C₂₄ lysophosphatidylcholine inthe membrane is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29% or 30% (w/w) of the amount of the at least one amphipathiclipid in the membrane.

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of the at least one amphipathic lipid in themembrane. In yet other embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1, 2, 3, 4, 5, 6, 7, 8,9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of the atleast one amphipathic lipid in the membrane.

In certain embodiments, the stabilizer, such as but not limited tom-cresol, is contacted with the membrane, and/or the lipid componentsthat assemble to form the membrane, at a (w/w) ratio ranging from about1:1 to about 1:30. In other embodiments, the stabilizer, such as but notlimited to m-cresol, is contacted with the membrane, and/or the lipidcomponents that assemble to form the membrane, at a (w/w) ratio of about1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5,1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27,1:28, 1:29 or 1:30.

In certain embodiments, the size of the nanoparticle ranges from about10 nm to about 150 nm. In other embodiments, the size of thenanoparticle is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm,80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or 150 nm.

In certain embodiments, a therapeutic agent (such as, but not limitedto, insulin) is dispersed within and/or adsorbed onto the nanoparticle.In other embodiments, the therapeutic agent is covalently bound to thenanoparticle. In yet other embodiments, the therapeutic agent is notcovalently bound to the nanoparticle.

In certain embodiments, the therapeutic agent comprises at least oneselected from the group consisting of insulin, insulin analogs, GLP-1agonist, amylin, interferon, parathyroid hormone, calcitonin, serotonin,serotonin agonist, serotonin reuptake inhibitor, human growth hormone,GIP, anti-GIP monoclonal antibody, metformin, bromocriptine, dopamine,glucagon, and GLP-1. In other embodiments, the therapeutic agent isinsulin.

In certain embodiments, the nanoparticle is suspended in an aqueoussolution comprising a free dissolved therapeutic agent that is notdispersed within the nanoparticle.

In certain embodiments, the nanoparticle-dispersed insulin and the freedissolved insulin are independently selected from the group consistingof insulin lispro, insulin aspart (including FIASP®, Novo Nordisk),regular insulin, insulin glargine, insulin zinc, extended human insulinzinc suspension, isophane insulin, human buffered regular insulin,insulin glulisine, recombinant human regular insulin, recombinant humaninsulin isophane, insulin detemir, biphasic human insulin, and insulindegludec (including TRESIBA®, Novo Nordisk).

In certain embodiments, the lipid further comprises cellulose acetatephthalate. In other embodiments, the cellulose acetate phthalate is atleast partially bound to the therapeutic agent dispersed within thenanoparticle.

In certain embodiments, at least one charged organic molecule is boundto the therapeutic agent dispersed within the nanoparticle. In otherembodiments, the charged organic molecule is at least one selected fromthe group consisting of protamines, polylysine, poly (arg-pro-thr)n in amole ratio of 1:1:1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6:1,histones, sugar polymers comprising a primary amino group,polynucleotides with primary amino groups, proteins comprising aminoacid residues with carboxyl (COO⁻) or sulfhydral (S⁻) functional groups,and acidic polymers (such as sugar polymers containing carboxyl groups).

In certain embodiments, the nanoparticle of the invention, andcompositions comprising the same, help deliver the therapeutic agentdispersed therewithin to the hepatocytes in the liver.

In certain embodiments, the compositions of the invention comprise aneffective dose of a hepatocyte targeted pharmaceutical composition thatcombines free therapeutic drug (such as, but not limited to, insulin)and therapeutic drug associated with the lipid-based nanoparticle of theinvention. The combination of free therapeutic drug and therapeutic drugassociated with the lipid-based nanoparticle creates a dynamicequilibrium process between the two forms of therapeutic drug thatoccurs in vivo to help control the movement of free therapeutic drug tothe receptor sites of hormonal action. In the case of insulin as thetherapeutic drug, those receptor sites are the muscle and adiposetissues of a diabetic patient. Hepatocyte targeted therapeutic drug isalso delivered to the liver of a patient over a different designatedtime period than free therapeutic drug, thereby introducing newpharmacodynamic profiles of therapeutic drug when the therapeutic drugremains associated with the nanoparticle and/or when free therapeuticdrug is released from the nanoparticle. In addition, a portion oftherapeutic drug that is associated with the nanoparticle is targeted tothe liver. In the case of insulin as the therapeutic drug, the newpharmacodynamic profile of the product provides not only basal insulinfor peripheral tissues, but also meal-time hepatic therapeutic drugstimulation for the management of hepatic glucose storage during a meal.Free insulin is released from the site of administration and isdistributed throughout the body. Insulin associated with the lipid-basednanoparticle is delivered to the liver. The rate of release of insulinassociated with the nanoparticle is different than the rate of releaseof free insulin from the site of administration. These different releaserates of insulin delivery, combined with the targeted delivery ofinsulin associated with the nanoparticle to the liver, provide for thenormalization of glucose concentrations in patients with Type 1 and Type2 diabetes mellitus, as well as patients with metabolic derangements,such as but not limited to metabolic syndrome with elevated insulinlevels, steatosis, and/or steatohepatitis. In certain embodiments, thehepatocyte targeted composition comprises any therapeutically effectiveinsulin or insulin derivative or analog, or any combination of two ormore types of insulin or insulin derivative or analog.

Compounds described herein also include isotopically labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, isotopically labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In other embodiments, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet otherembodiments, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

Compounds of the invention can in certain embodiments form acids orbases. In certain embodiments, the invention contemplates acid additionsalts. In other embodiments, the invention contemplates base additionsalts. In yet other embodiments, the invention contemplatespharmaceutically acceptable acid addition salts. In yet otherembodiments, the invention contemplates pharmaceutically acceptable baseaddition salts. Pharmaceutically acceptable salts refer to salts ofthose bases or acids that are not toxic or otherwise biologicallyundesirable.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids(including hydrogen phosphate and dihydrogen phosphate). Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which include formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic,glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium, lithium and copper, ironand zinc salts. Pharmaceutically acceptable base addition salts alsoinclude organic salts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. All ofthese salts may be prepared from the corresponding compound by reacting,for example, the appropriate acid or base with the compound.

Disclosed is a kit comprising any composition of the invention and aninstructional material which describes administering the composition toa tissue of a subject, such as a mammal. This kit may comprise a(preferably sterile) solvent suitable for dissolving or suspending thecomposition of the invention prior to administering the composition tothe subject, such as a mammal.

Methods

The invention provides methods of preparing the lipid-based nanoparticleof the invention. In certain embodiments, the method comprisescontacting in an aqueous system cholesterol, dicetyl phosphate,amphipathic lipid, and hepatocyte receptor binding molecule. In otherembodiments, the method comprises contacting in an aqueous systemcholesterol, dicetyl phosphate, amphipathic lipid, hepatocyte receptorbinding molecule, and at least one compound selected from the groupconsisting of a stabilizer and stearoyl lysophosphatidylcholine. In yetother embodiments, the method comprises contacting in an aqueous systemcholesterol, dicetyl phosphate, DSPC, and biotin-DHPE. In yet otherembodiments, the method comprises contacting in an aqueous systemcholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine,m-cresol, and biotin-DHPE.

In certain embodiments, the nanoparticle is formed in the absence of thetherapeutic agent, wherein optionally the nanoparticle is at leastpartially concentrated, purified or isolated, and wherein thetherapeutic agent is contacted with the nanoparticle, whereby at least aportion of the therapeutic agent is dispersed within the nanoparticle.

In certain embodiments, the composition is treated with celluloseacetate phthalate, which can bind non-covalently to at least a portionof the therapeutic agent dispersed within the nanoparticle and protectthe therapeutic agent from metabolic degradation. In other embodiments,the cellulose acetate phthalate is covalently bound to the therapeuticagent and/or any of the lipids that constitute the nanoparticle.

Further embodiments relating to certain methods for preparing and/orprocessing and/or purifying a nanoparticle can be found, for example, inU.S. Patent Application Nos. US20110135725 and US20090087479 and PCTPatent Application Publication No. WO 2018/169954, all of which areincorporated herein in their entireties by reference.

The invention further provides a method of treating a disease in amammal. In certain embodiments, the method comprises administering tothe mammal in need thereof a therapeutically effective amount of ananoparticle and/or a composition of the invention.

In certain embodiments, the disease is diabetes mellitus and thetherapeutic agent comprises insulin. In other embodiments, thetherapeutic agent further comprises a GLP-1 agonist and/or serotonin.

Administration/Dosage/Formulations

The invention also encompasses pharmaceutical compositions and methodsof their use. These pharmaceutical compositions may comprise an activeingredient (which can be one or more compositions of the invention, orpharmaceutically acceptable salts thereof) optionally in combinationwith one or more pharmaceutically acceptable agents. The compositionsset forth herein can be used alone or in combination with additionalcompounds to produce additive, complementary, or synergistic effects.

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder contemplatedherein. Further, several divided dosages, as well as staggered dosagesmay be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection, or may beadministered inhalationally, buccally and/or orally. Further, thedosages of the therapeutic formulations may be proportionally increasedor decreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder contemplated herein. An effective amountof the therapeutic compound necessary to achieve a therapeutic effectmay vary according to factors such as the state of the disease ordisorder in the patient; the age, sex, and weight of the patient; andthe ability of the therapeutic compound to treat a disease or disordercontemplated herein. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A non-limitingexample of an effective dose range for a therapeutic compound of theinvention is from about 1 and 5,000 mg/kg of body weight/per day. One ofordinary skill in the art would be able to study the relevant factorsand make the determination regarding the effective amount of thetherapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect, andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder contemplated herein.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, as long as the solvent or dispersion mediumdoes not disrupt the nanoparticle significantly. Prevention of theaction of microorganisms may be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it is preferableto include isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions may be brought aboutby including in the composition an agent that delays absorption, forexample, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 350 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial increments therebetween.

In certain embodiments, the dose of a compound and/or composition of theinvention is from about 1 mg and about 2,500 mg. In other embodiments, adose of a compound of the invention used in compositions describedherein is less than about 10,000 mg, or less than about 8,000 mg, orless than about 6,000 mg, or less than about 5,000 mg, or less thanabout 3,000 mg, or less than about 2,000 mg, or less than about 1,000mg, or less than about 500 mg, or less than about 200 mg, or less thanabout 50 mg. Similarly, in other embodiments, a dose of a secondcompound as described herein is less than about 1,000 mg, or less thanabout 800 mg, or less than about 600 mg, or less than about 500 mg, orless than about 400 mg, or less than about 300 mg, or less than about200 mg, or less than about 100 mg, or less than about 50 mg, or lessthan about 40 mg, or less than about 30 mg, or less than about 25 mg, orless than about 20 mg, or less than about 15 mg, or less than about 10mg, or less than about 5 mg, or less than about 2 mg, or less than about1 mg, or less than about 0.5 mg, and any and all whole or partialincrements thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound and/or composition of theinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the compound to treat, prevent, or reduce oneor more symptoms of a disease or disorder contemplated herein.

In certain embodiments, the container holds a lipid-based nanoparticle,which does not comprise a therapeutic agent of interest, such as but notlimited to an insulin or a derivative or analog thereof. In otherembodiments, the container holds a lipid-based nanoparticle, whichcomprises a therapeutic agent of interest, such as but not limited to aninsulin or a derivative or analog thereof. In yet other embodiments, thecontainer further holds a therapeutic agent of interest, such as but notlimited to an insulin or a derivative or analog thereof.

Illustrative Non-Limiting Methods of Treating

Patients with Type 1 or Type 2 diabetes mellitus, as well as patientswith metabolic derangements, such as but not limited to metabolicsyndrome with elevated insulin levels, steatosis, and/orsteatohepatitis, can be administered an effective amount of ananoparticle of the invention comprising an insulin. When thiscomposition is administered subcutaneously, a portion of the compositionenters the circulatory system where the composition is transported tothe liver and other areas. The extended amphipathic lipid binds thelipid construct to receptors of hepatocytes. A portion of theadministered composition is exposed to an external gradient in vivo,where insulin can be solubilized and then move from the lipid constructthereby supplying insulin to the muscle and adipose tissue. Insulin thatremains with the lipid construct maintains the capability of beingdirected to the hepatocyte binding receptor on the hepatocytes in theliver. Therefore, two forms of insulin are produced from this particularlipid construct. In an in vivo setting, free and lipid associatedinsulin are generated in a time-dependent manner.

Administration of the nanoparticles and compositions comprising same canbe through any of the accepted modes of administration for insulin thatare desired to be administered. These methods include oral, parenteral,nasal and other systemic or aerosol forms. These methods further includepump delivery systems.

Oral administration of a nanoparticle of the invention is followed byintestinal absorption of insulin associated with the nanoparticle of theinvention into the circulatory system of the body, where it is alsoexposed to the physiological pH of the blood. The nanoparticle istargeted for delivery to the liver and may be shielded by the presenceof cellulose acetate phthalate within the nanoparticle of the invention.In the case of oral administration, the shielded nanoparticletransverses the oral cavity, migrates through the stomach and moves intothe small intestine, where the alkaline pH of the small intestinedegrades the cellulose acetate phthalate shield. The deshieldednanoparticle is absorbed into the circulatory system. This enables thenanoparticle to be delivered to the sinusoids of the liver. A receptorbinding molecule, such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) or anyother hepatocyte specific molecule, provides a means for lipid constructto bind to the receptor and then be engulfed or endocytosed by thehepatocytes. Insulin is then released from the nanoparticle where, upongaining access to the cellular environment, it performs its designatedfunction with regard to acting as an agent to control diabetes mellitus.

Patients with Type 1 or Type 2 diabetes mellitus, as well as patientswith metabolic derangements, such as but not limited to metabolicsyndrome with elevated insulin levels, steatosis, and/orsteatohepatitis, may be administered an effective amount of ananoparticle comprising a mixture of free glargine insulin and glargineinsulin associated with the nanoparticle. Glargine insulin can becombined with other forms of insulin, such as insulin lispro, insulinaspart (including FIASP®, Novo Nordisk), regular insulin, insulinglargine, insulin zinc, extended human insulin zinc suspension, isophaneinsulin, human buffered regular insulin, insulin glulisine, recombinanthuman regular insulin, recombinant human insulin isophane, insulindetemir, biphasic human insulin, and insulin degludec (includingTRESIBA®, Novo Nordisk) or premixed combinations of any of theaforementioned insulins, a derivative thereof, and a combination of anyof the aforementioned insulins. The composition can be administered by asubcutaneous or oral route.

After a composition is administered to a patient by subcutaneousinjection, the in situ physiological environment in the injection area,the morphology and chemical structures of free insulin and the insulinassociated with the nanoparticle begin to change. For example, as the pHof the environment around the free glargine insulin and the glargineinsulin associated with the nanoparticle increases after being dilutedwith physiological media, the pH reaches the isoelectric point ofglargine insulin, where flocculation, aggregation and precipitationreactions occur for both free glargine insulin and glargine insulinassociated with the nanoparticle. In certain embodiments, free glargineinsulin changes from a soluble form at injection, to a insoluble form ata pH near its isoelectric point of pH 5.8-6.2, and then to a solubleform at physiological pH. The rates at which these processes occurdiffer between free glargine insulin and glargine insulin associatedwith the nanoparticle. The free glargine insulin is directly exposed tochanges in pH and dilution. Exposure of glargine insulin associated withthe nanoparticle to small changes in pH and dilution at physiological pHis delayed due to the time required for diffusion of physiologicalfluids or media through the lipid bilayer in the nanoparticle. The delayin the release of insulin from the lipid construct as well as the delayof the release of the insulin associated with the nanoparticle is afeature of the invention since it affects and augments the biologicaland pharmacological response in vivo.

Oral administration of a pharmaceutical composition that combines freeglargine insulin and glargine insulin associated with a nanoparticle isfollowed by intestinal absorption of glargine insulin associated withthe nanoparticle into the circulatory system of the body, where it isalso exposed to the physiological pH of the blood. In certainembodiments, the composition comprises a delayed release matrix whichreleases HDV glargine over a prolonged period of time, in order toachieve a 24-hour dose regimen. All or a portion of the nanoparticle isdelivered to the liver.

Patients with Type 1 or Type 2 diabetes mellitus, as well as patientswith metabolic derangements, such as but not limited to metabolicsyndrome with elevated insulin levels, steatosis, and/orsteatohepatitis, can be administered an effective amount of a hepatocytetargeted composition comprising a mixture of free recombinant humaninsulin isophane (NPH) plus free recombinant human regular insulin alongwith recombinant human insulin isophane and recombinant human regularinsulin which are both associated with a nanoparticle. Recombinant humaninsulin isophane can be combined with other forms of insulin, suchinsulin lispro, insulin aspart (including FIASP®, Novo Nordisk), regularinsulin, insulin glargine, insulin zinc, extended human insulin zincsuspension, isophane insulin, human buffered regular insulin, insulinglulisine, recombinant human regular insulin, recombinant human insulinisophane, insulin detemir, biphasic human insulin, and insulin degludec(including TRESIBA®, Novo Nordisk, ultralong-acting basal insulinanalogue; has one single amino acid deleted in comparison to humaninsulin, and is conjugated to hexadecanedioic acid via gamma-L-glutamylspacer at the amino acid lysine at position B29), or any (premixed)combinations thereof.

In certain embodiments, the composition comprises a delayed releasematrix which releases HDV NPH over a prolonged period of time, in orderto achieve a 24-hour dose regimen.

Oral administration of a pharmaceutical composition that combines freerecombinant human insulin isophane and recombinant human insulinisophane associated with a nanoparticle is followed by intestinalabsorption of recombinant human insulin isophane associated with thenanoparticle into the circulatory system of the body where it is alsoexposed to the physiological pH of the blood. All or a portion of thenanoparticle is delivered to the liver, while the non-HDV isophane isslowly absorbed from a slow release matrix for release into the generalcirculation.

As the physiological dilution is increased in situ in the subcutaneousspace or upon entering into the circulatory system, free recombinanthuman insulin isophane and recombinant human insulin isophane associatedwith the nanoparticle encounter a normal physiological pH environment ofpH 7.4. As a result of dilution free recombinant human insulin isophanechanges from an insoluble form at injection, to a soluble form atphysiological pH. In the soluble form, recombinant human insulinisophane migrates through the body to sites where it is capable ofeliciting a pharmacological response. Recombinant human insulin isophaneassociated with the nanoparticle becomes solubilized and released fromthe nanoparticle at a different rate that is slower than that of freerecombinant human insulin isophane. This is because recombinant humaninsulin isophane associated with the nanoparticle has to traverse thecore volume and lipid domains of the nanoparticle before it contacts thebulk phase media.

The amount of insulin administered will be dependent on the subjectbeing treated, the type and severity of the affliction, the manner ofadministration and the judgment of the prescribing physician. Althougheffective dosage ranges for specific biologically active substances ofinterest are dependent upon a variety of factors and are generally knownto one of ordinary skill in the art, some dosage guidelines can begenerally defined. For most forms of administration, the nanoparticlewill be suspended in an aqueous solution and generally not exceed 4.0%(w/v) of the total formulation. The drug component of the formulationwill in certain embodiments be less than 20% (w/v) of the formulationand generally greater than 0.01% (w/v).

In certain embodiments, the pharmaceutical composition comprises HDVinsulin, and no free insulin. In such cases, all of the insulin withinthe composition is targeted to the liver. In other embodiments, thepharmaceutical composition comprises HDV insulin and free insulin(non-HDV insulin). The ratio between HDV insulin and free insulin canbe, in non-limiting example, about 0.1:99.9, 0.2:99.8, 0.3:99.7,0.4:99.6, 0.5:99.5, 0.6:99.4, 0.7:99.3, 0.8:99.2, 0.9:99.1, 1:99, 2:98,3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 12:88, 14:86, 16:84,18:82, 20:80, 22:78, 24:76, 25:75, 26:74, 28:72, 30:70, 32:68, 34:66,36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, and/or 50:50.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 5% with the balance made up from non-toxic carriers can beprepared.

The exact composition of these formulations may vary widely depending onthe particular properties of the drug in question. In certainembodiments, they comprise from 0.01% to 5%, and preferably from 0.05%to 1% active ingredient for highly potent drugs, and from 2%-4% formoderately active drugs.

The percentage of active ingredient contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the activity of the active ingredient and the needs of the subject.However, percentages of active ingredient of 0.01% to 5% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. In certainembodiments, the composition comprises 0.2%-2.0% of the active agent insolution.

Administration

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds and/or compositions for use in theinvention may be formulated for administration by any suitable route,such as for oral or parenteral, for example, transdermal, transmucosal(e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal(e.g., trans- and perivaginally), (intra)nasal and (trans)rectal),intravesical, intrapulmonary, intraduodenal, intragastrical,intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial,intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds and/or compositions of theinvention may be in the form of tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Parenteral Administration

For parenteral administration, the compounds and/or compositions of theinvention may be formulated for injection or infusion, for example,intravenous, intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Pulmonary Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 microns, and preferably from about 1 to about6 microns. Such compositions are conveniently in the form of dry powdersfor administration using a device comprising a dry powder reservoir towhich a stream of propellant may be directed to disperse the powder orusing a self-propelling solvent/powder-dispensing container such as adevice comprising the active ingredient dissolved or suspended in alow-boiling propellant in a sealed container. Preferably, such powderscomprise particles wherein at least 98% of the particles by weight havea diameter greater than 0.5 microns and at least 95% of the particles bynumber have a diameter less than 7 microns. More preferably, at least95% of the particles by weight have a diameter greater than 1 nanometerand at least 90% of the particles by number have a diameter less than 6microns. Dry powder compositions preferably include a solid fine powderdiluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile for administration by injection,comprising the active ingredient, and may conveniently be administeredusing any nebulization or atomization device. In certain embodiments,the compounds and/or compositions of the invention are sterile filteredbefore administration to the subject. Such formulations may furthercomprise one or more additional ingredients including, but not limitedto, a flavoring agent such as saccharin sodium, a volatile oil, abuffering agent, a surface active agent, or a preservative such asmethylhydroxybenzoate. The droplets provided by this route ofadministration preferably have an average diameter in the range fromabout 0.1 to about 200 microns.

Intranasal Delivery

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 microns. Such a formulation is administered in themanner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 75% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compositions may be formulated with asuitable polymer or hydrophobic material that provides sustained releaseproperties to the compounds and/or compositions. As such, thecompositions and/or compositions for use the method of the invention maybe administered in the form of microparticles, for example, by injectionor in the form of wafers or discs by implantation.

In certain embodiments, the compounds and/or compositions of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound and/orcomposition of the present invention depends on the age, sex and weightof the patient, the current medical condition of the patient and theprogression of a disease or disorder contemplated herein in the patientbeing treated. The skilled artisan is able to determine appropriatedosages depending on these and other factors.

A suitable dose of a compound and/or composition of the presentinvention may be in the range of from about 0.01 mg to about 5,000 mgper day, such as from about 0.1 mg to about 1,000 mg, for example, fromabout 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.The dose may be administered in a single dosage or in multiple dosages,for example from 1 to 4 or more times per day. When multiple dosages areused, the amount of each dosage may be the same or different. Forexample, a dose of 1 mg per day may be administered as two 0.5 mg doses,with about a 12-hour interval between doses.

It is understood that the amount of compound and/or composition dosedper day may be administered, in non-limiting examples, every day, everyother day, every 2 days, every 3 days, every 4 days, or every 5 days.For example, with every other day administration, a 5 mg per day dosemay be initiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. Incertain embodiments, patients require intermittent treatment on along-term basis upon any recurrence of symptoms and/or infection.

The compounds and/or compositions for use in the method of the inventionmay be formulated in unit dosage form. The term “unit dosage form”refers to physically discrete units suitable as unitary dosage forpatients undergoing treatment, with each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, optionally in association with a suitablepharmaceutical carrier. The unit dosage form may be for a single dailydose or one of multiple daily doses (e.g., about 1 to 4 or more timesper day). When multiple daily doses are used, the unit dosage form maybe the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds and/orcompositions lies preferably within a range of circulatingconcentrations that include the ED₅₀ with minimal toxicity. The dosageoptionally varies within this range depending upon the dosage formemployed and the route of administration utilized.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Generally, thenomenclature used herein and the laboratory procedures in organicchemistry and protein chemistry are those well known and commonlyemployed in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “A1c” or “A1c” or “HbA1c” or “hemoglobin A1c” or “HBA1c” or“HgbA1c” or “haemoglobin A1c” or “HbA1c” or “Hb1c” refers to a form ofhemoglobin that is covalently bound to glucose. A1c is formed in anon-enzymatic glycation pathway by hemoglobin's exposure to plasmaglucose. A1c is measured primarily to identify the three-month averageplasma glucose concentration, and thus can be used as a diagnostic testfor diabetes and as assessment test for glycemic control in people withdiabetes. The ratio of A1c to total hemoglobin (% A1c) (generallymeasured as mass/mass) is used to diagnose diabetes (according to 1993Diabetes Control and Complications Trial or DCCT): normal individualshave less than 5.7% A1, pre-diabetic individuals have 5-7-6.4% A1c, anddiabetic individuals have greater than 6.5% A1c. The DCCT % A1c valuecan be converted to the International Federation of Clinical Chemistryand Laboratory Medicine (IFCC) units using the formula:

IFCC HbA1c(mmol/mol)=[DCCT HbA1c(%)−2.14]×10.929

As used herein, the term “about” is understood by persons of ordinaryskill in the art and varies to some extent on the context in which it isused. As used herein when referring to a measurable value such as anamount, a temporal duration, and the like, the term “about” is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, the term “active ingredient” refers to a therapeuticagent that is to be delivered to a subject to produce a therapeuticeffect in the subject. Non-limiting examples of active ingredientscontemplated within the invention are insulin, interferon, parathyroidhormone, calcitonin, serotonin, serotonin agonist, serotonin reuptakeinhibitor, human growth hormone, GIP, anti-GIP monoclonal antibody,metformin, bromocriptine, dopamine, glucagon and/or GLP-1.

The term “amphipathic lipid” means a lipid molecule having a polar andnon-polar end.

By “aqueous media” is meant water or water containing buffer or salt.

As used herein, the term “basal insulin” or “background insulin” isinsulin that is taken to keep blood glucose levels at consistent levelsduring periods of fasting. Basal insulin is thus needed to keep bloodglucose levels under control, and to allow the cells to take in glucosefor energy. Basal insulin is usually taken once or twice a day dependingon the insulin. Basal insulin needs to act over a relatively long periodof time, and thus is either long acting insulin or intermediate insulin.

As used herein, the term “basal glucose control” refers to the glucosecontrol that is afforded by use of basal insulin, or an equivalentthereof.

The term “bioavailability” refers to a measurement of the rate andextent that insulin reaches the systemic circulation and is available atthe sites of action.

As used herein, the term “bolus insulin” refers to insulin that isspecifically taken just before, at, or just after meal times to keepblood glucose levels under control following a meal. Bolus insulin needsto act quickly and is generally short acting insulin or rapid actinginsulin.

As used herein, the term “bolus glucose control” refers to the glucosecontrol that is afforded by use of bolus insulin, or an equivalentthereof.

As used herein, the term “CGM” refers to continuous glucose monitoring.

In one aspect, the terms “co-administered” and “co-administration” asrelating to a subject to refer to administering to the subject acompound of the invention or salt thereof along with a compound that mayalso treat any disease or disorder contemplated herein and/or with acompound that is useful in treating other medical conditions but whichin themselves may cause or facilitate any disease or disordercontemplated herein. In certain embodiments, the co-administeredcompounds are administered separately, or in any kind of combination aspart of a single therapeutic approach. The co-administered compound maybe formulated in any kind of combinations as mixtures of solids andliquids under a variety of solid, gel, and liquid formulations, and as asolution.

As used herein, a “disease” is a state of health of a subject whereinthe subject cannot maintain homeostasis, and wherein if the disease isnot ameliorated then the subject's health continues to deteriorate.

As used herein, a “disorder” in a subject is a state of health in whichthe subject is able to maintain homeostasis, but in which the subject'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the subject's state of health.

As used herein, the term “ED₅₀” refers to the effective dose of aformulation that produces 50% of the maximal effect in subjects that areadministered that formulation.

As used herein, an “effective amount,” “therapeutically effectiveamount” or “pharmaceutically effective amount” of a compound is thatamount of compound that is sufficient to provide a beneficial effect tothe subject to which the compound is administered.

The term “free active ingredient” or “free therapeutic agent” refers toan active ingredient or therapeutic agent that is not dispersed withinthe lipid particle (i.e., located within, adsorbed on and/or bound tothe lipid particle membrane).

The terms “glargine” and “glargine insulin” both refer to a recombinanthuman insulin analog which differs from human insulin in that the aminoacid asparagine at position A21 is replaced by glycine and two argininesare added to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

As used herein, the term “hyperinsulinemia” refers to a condition inwhich there are excess levels of insulin circulating in the bloodrelative to the level of glucose. Hyperinsulinemia can be an unwantedside effect of administration of exogenous insulin to a diabetic patient(thus being a form of iatrogenic hyperinsulinemia; see Cryer, 2008,Diabetes 57(12):3169-76, McCrinson & Sherwin, 2010, Diabetes59(10):2333-9; Wang, et al., 2013, J. Diab. & Its Compl. 27(1):70-74;all of which are incorporated herein in their entireties by reference).That condition can trigger complications such as metabolic disease,hypoglycemia, increased risk of polycystic ovary syndrome (PCOS),increased synthesis of VLDL (hypertriglyceridemia), hypertension(insulin increases sodium retention by the renal tubules), coronaryartery disease (CAD; increased insulin damages endothelial cells),increased risk of cardiovascular disease, and/or weight gain andlethargy.

As used herein, the term “hypoglycemic event” or “hypoglycemia event”refers to an event wherein the subject's blood sugar is lower than 70mg/dL for a significant amount of time, such as but not limited to 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certainembodiments, a hypoglycemic event is defined as a series of CGM valuesless than about 54 mg/dL, separated by 20 min or more, with nointervening values of 54 mg/dL or more. In certain embodiments, ahypoglycemic event is defined as over 15 min of CGM values less thanabout 54 mg/dL.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat can be used to communicate the usefulness of the composition and/orcompound of the invention in a kit. The instructional material of thekit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

The term “insulin” refers to natural or recombinant forms of insulin,and derivatives of the aforementioned insulins. Examples of insulininclude, but are not limited to insulin lispro (such as, for example,ADMELOG®, Sanofi), insulin aspart (such as, for example, FIASP®, NovoNordisk), regular insulin, insulin glargine (such as, for example,BASAGLAR®, Lilly), insulin zinc, human insulin zinc extended, isophaneinsulin, human buffered regular insulin, insulin glulisine, recombinanthuman regular insulin, recombinant human insulin isophane, insulindetemir, biphasic human insulin, and insulin degludec (includingTRESIBA®, Novo Nordisk, ultralong-acting basal insulin analogue; has onesingle amino acid deleted in comparison to human insulin, and isconjugated to hexadecanedioic acid via gamma-L-glutamyl spacer at theamino acid lysine at position B29). Also included are animal insulins,such as bovine or porcine insulin.

As used herein, the term “iotrogenic” refers to any illness caused by amedical examination or treatment.

The term “isoelectric point” refers to the pH at which theconcentrations of positive and negative charges on the protein are equaland, as a result, the protein will express a net zero charge. At theisoelectric point, a protein will exist almost entirely in the form of azwitterion, or hybrid between forms of the protein. Proteins are leaststable at their isoelectric points, and are more easily coagulated orprecipitated at this pH. However, proteins are not denatured uponisoelectric precipitation since this process is essentially reversible.

The term “lipid construct” refers to a lipid and/or phospholipidparticle in which individual lipid molecules interact to create abipolar lipid membrane that defines the boundaries of the lipidconstruct.

As the term is used herein, “to modulate” or “modulation of a biologicalor chemical process or state refers to the alteration of the normalcourse of the biological or chemical process, or changing the state ofthe biological or chemical process to a new state that is different thanthe present state. For example, modulation of the isoelectric point of apolypeptide may involve a change that increases the isoelectric point ofthe polypeptide. Alternatively, modulation of the isoelectric point of apolypeptide may involve a change that decreases the isoelectric point ofa polypeptide.

As used herein, a “metabolic derangement” refers to a metabolic disorderor disease relating to uncontrolled, elevated, or fluctuating insulinlevels, such as but not limited to metabolic syndrome with elevatedinsulin levels, steatosis, and/or steatohepatitis.

The term “non-glargine insulin” refers at all insulins, either naturalor recombinant that are not glargine insulin. The term includesinsulin-like moieties, including fragments of insulin molecules, thathave biological activity of insulins.

As used herein, the term “pharmaceutical composition” or “composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a subject.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound useful within theinvention, and is relatively non-toxic, i.e., the material may beadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with any of the components of thecomposition in which it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the subject such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the subject. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the subject.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof.

The term “prevent,” “preventing” or “prevention,” as used herein, meansavoiding or delaying the onset of symptoms associated with a disease orcondition in a subject that has not developed such symptoms at the timethe administering of an agent or compound commences.

Disease, condition and disorder are used interchangeably herein.

By the term “specifically bind” or “specifically binds,” as used herein,is meant that a first molecule preferentially binds to a second molecule(e.g., a particular receptor or enzyme), but does not necessarily bindonly to that second molecule.

As used herein, a “subject” may be a human or non-human mammal or abird. Non-human mammals include, for example, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals. In certainembodiments, the subject is human.

The term “treat,” “treating” or “treatment,” as used herein, meansreducing the frequency or severity with which symptoms of a disease orcondition are experienced by a subject by virtue of administering anagent or compound to the subject.

The term “well controlled diabetes” refers to a diabetic or pre-diabeticsubject that receives treatment that allows for keeping fasting bloodsugars below 140 mg/dL. In certain embodiments, the fasting blood sugarsthreshold is below 140 mg/dL, below 130 mg/dL, below 120 mg/dL, below110 mg/dL, or below 100 mg/dL. In certain embodiments, the fasting bloodsugars range is 70-120 mg/dL. In certain embodiments, the fasting bloodsugars range is 80-100 mg/dL. In certain embodiments, the fasting bloodsugars range is 70-120 mg/dL. In certain embodiments, the fasting bloodsugars range is 70-100 mg/dL.

Throughout this disclosure, various aspects of the invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range and, when appropriate,partial integers of the numerical values within ranges. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of this inventionand covered by the claims appended hereto. For example, it should beunderstood, that modifications in reaction conditions, including but notlimited to reaction times, reaction size/volume, and experimentalreagents, such as solvents, catalysts, pressures, atmosphericconditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents,with art-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, point out specific embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

The materials and methods used in the experiments presented in thisExperimental Example are now described.

Example 1: Divergent Hypoglycemic Effects of Hepatic Directed PrandialInsulin—A Six-Month Study in Type 1 Diabetes Mellitus (T1DM)

In one aspect, HDV-I is an insulin-agnostic delivery system thatutilizes a biotin-containing lipid (such as but not limited tobiotin-phosphatidylethanolamine) in a phospholipid matrix, targetinginsulin to the liver. Mimicking portal vein delivery, subcutaneous (SC)injection of HDV-I provides a more physiologic treatment paradigm.Treatment with SC HDV-human regular insulin (RHI) reduces postprandialglucose excursions compared to SC RHI. Without wishing to be limited byany theory, HDV-I's flat dose-response effect on hepatic glucose balancein preclinical studies supports a fixed combination for treatment.

In the present study the use of HDV-insulin lispro (HDV-L) vs. insulinlispro (LIS) in treating type 1 diabetes mellitus (T1DM) was assessed.HDV-L in this study contained 1% HDV-bound LIS and 99% unbound LIS.

ISLE-1 was a 26-week, Phase 2b, multicenter, randomized, double-blind,non-inferiority trial. Among 176 randomized patients (HDV-L, n=118; LIS,n=58), difference in change from baseline A1c at Week 26 was +0.09% (95%CI −0.18% to 0.35%), confirming non-inferiority (pre-specified margin0.4%). Baseline A1c modified the treatment group effect on hypoglycemiarisk (interaction p-value <0.001), with less risk of hypoglycemia (andlower insulin dosing with similar A1c outcome) with HDV-L compared toLIS at higher A1c, but opposite hypoglycemia effects at lower A1c(despite similar A1c and insulin dosing). No safety signals wereidentified. The present results indicate that HDV-L's hepaticbiodistribution appears to potentiate insulin effect in T1DM.

a. Design and Methods

Design and Participants:

ISLE-1 was a 26-week, Phase 2b, multicenter, randomized, double-blind,trial in T1DM treated with multiple daily injections (MDI) of insulin.The primary objective was A1c non-inferiority with 26 weeks of HDV-Lversus LIS.

Main inclusion criteria were: age ≥18 years; T1D for ≥12 months; A1c≥7.0 (≥58 mmol/mol) to ≤10.5% (≤91 mmol/mol); treated with insulinsglargine or detemir for basal coverage. Main exclusion criteria weretotal insulin dose ≥1.5 IU/kg/day or NPH insulin as basal.

Procedures:

Participants were randomized 2:1 ([HDV-L:LIS), stratified by screeningA1c (<8.5% [69 mmol/L] vs. ≥8.5%). Study medications were HDV-L (0.8 mlHDV solution in 10 ml commercial LIS) and comparator, LIS (comparablydiluted with water).

Prandial dosing of HDV-lispro or control lispro was 15 minutes prior tothe meal, and basal insulins were administered either as a single dailydose or a divided twice per day dosing, every 12 hours.

Informed of ˜10% dilution, participants continued their current insulinparameters. Hypoglycemia was recorded on Case Report Forms (CRFs) basedon subject diaries and SMBG records, subjectively investigator-judged as“mild,” “moderate,” “severe,” or “life threatening.” Blinded continuousglucose monitoring (CGM) (Dexcom. G4) was used for 5-7 days to assessglucose at baseline, weeks 13 and 26. A1c, lipids, and liver enzymeswere measured approximately monthly. Liver fat content MRIs wereperformed in a subset.

Statistical Analysis:

The intent to treat (ITT) population included all randomized subjectsreceiving at least one dose of study treatment. Safety analyses includedall randomized subjects. A sample size of 150 with assumed A1c SD of0.8% and assumed A1c treatment difference of 0.4% had 99.9% power fornon-inferiority pre-specified 0.4% margin. Mean A1c change was analyzedusing ANCOVA within intent-to-treat (ITT) cohort at each visit. Post hocsubgroup analyses (baseline A1c <8.5% vs ≥8.5%) were performed, this cutpoint corresponding to pre-specified randomization strata. Directlikelihood models were used for treatment arm A1c comparisons, % time<54 mg/dL, bolus insulin, and basal insulin within the two A1csubgroups. Poisson regression models adjusting for site as random effectcompared “severe” hypoglycemia incidence rates within A1c groups,testing for baseline A1c by treatment group interaction. Eventnumber/subject was truncated at 15, accounting for extreme outliers.

b. Discussion

Subjects were randomly assigned HDV-L (n=118) or LIS (n=58). 62% ofHDV-L patients were male, with 72% of LIS male. Mean (±SD) baseline agewas 46.7±14.4 (HDV-L) and 44.1±15.7 (LIS). Mean (±SD) baseline HbA1c was8.12±0.79 (HDV-L) and 8.22±0.90 (LIS).

Mean change in A1c baseline to Week 26 was −0.09% with (HDV-L) and−0.16% (LIS), (estimated treatment difference [ETD], HDV-L-LIS: +0.09%[95% CI −0.18 to 0.35]), confirming HDV-L non-inferiority. Analysis ofhypoglycemia outcomes showed that baseline A1c status modified thetreatment group effect on “severe” hypoglycemia incidence (p-value forinteraction <0.001), with less hypoglycemia in HDV-L compared to LISwith poor control but higher risk in HDV-L with better control.

Further analyses were based on subgroups (A1c ≥8.5% vs. <8.5%). HDV-Ltreated subjects with baseline A1c ≥8.5% showed a CRF-reported incidencerate of “severe” hypoglycemia significantly lower than LIS (69 vs. 97events/100 person-years, p=0.03), and their percentage time <54 mg/dLduring Week 26 (FIG. 1A) showed trend for reduction (median 0.7% vs.2.6% for HDV-L and LIS, respectively, p=0.09). Conversely, with baselineA1c <8.5%, CRFs reported higher incidence of “severe” hypoglycemia withHDV-L than LIS (191 vs. 21, p=0.001), and time <54 mg/dL during Week 26(FIG. 1B) trended higher (median 2.0% vs. 0.6%, p=0.16). No “lifethreatening” events were recorded.

Exploring these divergent hypoglycemia findings, insulin dosing wasanalyzed. Subjects with A1c ≥8.5% showed similar A1c reductions for bothtreatments at Week 26 (p=0.35) (FIG. 1C). However, HDV-Ltreated-subjects achieved A1c reductions with ˜25% less bolus insulinthan LIS subjects (mean 0.29 U*kg⁻¹*day⁻¹ vs. 0.38, respectively,p=0.02), with comparable basal doses (mean 0.38 U*kg⁻¹*day⁻¹ vs. 0.45,respectively, p=0.37) at study end (FIG. 1E). HDV-L and LIS subjectswith baseline A1c <8.5% both showed little change in A1c over time (FIG.1D) without difference in bolus/basal insulin dosage at endpoint (p=0.86and 0.90 for basal and bolus, respectively) (FIG. 1F).

Lipids remained mostly stable throughout study; however, a significantreduction in total cholesterol with HDV-L (−6.5 mg/dL) vs. LIS (7.3mg/dL) was observed (ETD: HDV-L-LIS: −12.0 mg/dL [95% CI−21.1 to −2.9,p=0.01). Liver function tests at Weeks 5 and 19 showed stable ALT/ASTand bilirubin levels for both treatments. Of 21 subjects studied withMM, 4 had measurable baseline liver fat; one subject (treated withHDV-L) showed measurable liver fat increase (3.1% baseline; 11.4%endpoint), without other evidence of hepatic dysfunction. Notreatment-related serious adverse events were reported.

This is the first six-month study to demonstrate efficacy and safety ofa liver-targeting rapid-acting insulin formulation in T1DM. HDV-L wasnon-inferior to LIS by change in A1c, with significant total cholesterolreduction and no treatment-related severe adverse events. In contrast topeglispro safety results (Jacober, et al., 2016, Diabetes Obes Metab. 18(Suppl 2):3-16), the present study showed no between-group difference inALT.

In certain embodiments, administration of HDV-L provides morephysiologic insulin distribution than free insulin administration. Inother embodiments, by delivering a portion of the SC dose directly tothe liver, ˜30-60% of oral carbohydrate is sequestered as hepaticglycogen, reducing peripheral glucose exposure and demanding reductionin peripheral insulin exposure.

Without wishing to be limited by any theory, less well controlled HDV-Lsubjects did not meaningfully alter HDV-L doses over time (whereas LISwas increased by ˜25%) yet experienced less CRF-reported severehypoglycemia and less time <54 mg/dL as compared to LIS, withoutdifference in A1c between or within treatments. Without wishing to belimited by any theory, better-controlled HDV-L subjects failed torecognize a functional increase in insulin potency, resulting in a trendfor increased time spent <54 mg/dL and significant increase inCRF-reported hypoglycemia, despite no difference in their insulin dosingor A1c outcomes. The strikingly divergent hypoglycemia risk findings anddiffering insulin dose adjustments observed in poor-versusbetter-controlled subgroups can be unified by the hypothesis that, byaltering biodistribution of SC insulin to better include the liver, HDVincreases the functional potency of insulin in both high- and lower-A1csubgroups.

A downstream consequence of increased glycogen storage should beimproved availability of hepatic glucose to counteract hypoglycemia;this may have occurred with HDV-L at baseline A1c ≥8.5%, showing bothrelative (compared to LIS) and absolute reductions in time below 54mg/dL (FIG. 1A). In contrast, the lower A1c subgroup was apparentlyover-insulinized owing to the increased functional potency of HDV-L andlacked hyperglycemic “buffer” to limit absolute hypoglycemic risk.

The present results indicate that of HDV-L is non-inferior to LIS andits liver-targeted component potentiates insulin effect. HDV, when addedto lispro insulin, distributes meal time glucose to the liver and as aresult lowers peripheral blood glucose. In poorly controlled T1Dsubjects with HbA1c >8.5%, better glycemic control and reducedhypoglycemia was observed, even with lowered HDV-lispro insulin dosesover the course of the study. However, in better controlled subjects,those with HbA1c <8.5%, the reduction in peripheral glucose load led toincreased hypoglycemia incidence and severity, believed to be due to thepatients not reducing their basal (non HDV) insulin dose. In certainembodiments, addition of HDV to an insulin makes the insulin appear tobe more potent, necessitating a re-evaluation of the relationship ofmealtime (HDV-lispro) to basal insulin dosing, which covers times offasting, especially overnight.

Table 1 summarizes continuous glucose monitoring results in the Good toGreat Hypoglycemia study, in terms of increased HDV-related hypoglycemiaevents.

TABLE 1 Hypo- Treatment glycemia Difference median HDV − (quartiles)Baseline Endpoint Lispro p-val* % Time <70 12.0% 5.8% 8.3% 5.6% +3.4%0.01 mg/dL (4.5%, (3.1%, (6.6%, (3.3%, (+0.7% 16.0%) 10.7%) 10.6%) 8.4%)to +6.0%) % Time <54 4.4% 2.1% 3.3% 1.7% +1.4% 0.04 mg/dL (2.5%, (0.6%,(2.0%, (0.8%, (+0.1% 8.2%) 4.1%) 5.7%) 3.3%) to +3.0%) Area above 1.70.9 1.2 0.7 +0.5 0.02 curve <70 (0.9, (0.3, (0.9, (0.3, (+0.1 mg/dL 2.7)1.6) 1.9) 1.3) to +1.0) Area above 0.4 0.1 0.3 0.1 +0.1 0.07 curve <54(0.3, (0.0, (0.2, (0.1, (−0.01 mg/dL 0.8) 0.3) 0.6) 0.3) to +0.3) *Basedon a direct likelihood model adjusting for baseline value and a randomsite effect.

Example 2: Exploratory Randomized Open-Label 2-Arm Comparison ofDifferent Insulin Dosing Algorithms using Hepatic Directed Vesicle(HDV)-Insulin Lispro and Insulin Degludec to Determine Optimum BasalInsulin Dosing Regimens

The current standard of care for diabetes treatment comprises 1:1 dosesof bolus insulin and basal insulin. The present study aims to explorethe possibility of varying the ratio of HDV-containing bolus insulin andbasal insulin, so as to identify a dosing algorithm that allows for goodcontrol of blood glucose levels without causing hypoglycemia.

The present study is an open-label, multiple dose safety, tolerability,and efficacy study. The study subjects are afflicted with Type Idiabetes mellitus. There is a run-in phase where all subjects receiveInsulin Lispro (HUMALOG®) for 8 weeks and then are randomized to twogroups receiving HDV-formulated Insulin Lispro+Insulin Degludec dose.

In certain embodiments, the subject in one group receives a dose ofInsulin Degludec that is about 10% lower than the conventional dose ofInsulin Degludec used in diabetes treatments (which would have been thesame dose as the bolus insulin received under the 1:1 paradigm).

In certain embodiments, the subject in another group receives a dose ofInsulin Degludec that is about 40% lower than the conventional dose ofInsulin Degludec used in diabetes treatments (which would have been thesame dose as the bolus insulin received under the 1:1 paradigm).

In certain embodiments, HDV-insulin enables hepatic metabolism ofingested carbohydrate (glucose), reducing the glucose load to peripheraltissues, thus requiring an adjustment of basal doses of insulin so thatfasting hypoglycemia is reduced or eliminated. The present inventionprovides, in one aspect, a new, physiologically adjusted ratio ofmeal-time bolus HDV-insulin dose to the 24-hour basal insulin, such asbut not limited to degludec.

Inclusion Criteria:

-   -   1. Male or female of age 18 to 65 years, inclusive.    -   2. TIDM≥12 months    -   3. C-peptide <0.6 ng/mL (single retest allowed)    -   4. Treatment with rapid analog insulin for the previous 6 months    -   5. Not using insulin pump delivery systems during the previous 2        months    -   6. Use of personal continuous glucose monitoring (CGM)        technology for three months prior to starting study and        willingness to continue its use throughout study    -   7. BMI ≥18.0 kg/m² and ≤33.0 kg/m² 10.6.5%≤A1c≤8.5%

The present study comprises two arms, following a 3 month run-in periodwhere all subjects are brought to standard of care with insulin lisprowithout HDV with full characterization of their metabolic statusincluding HbA1c, and incidence and severity of hypoglycemia. The firstarm comprises (a) HDV-Insulin Lispro+Insulin Degludec dose reduced by40%. The second arm comprises (b) HDV-Insulin Lispro+Insulin Degludecdose reduced by 10%. The primary outcome measure comprises basal, bolus,and total insulin doses and basal/bolus ratios during the last 2 weeksof the treatment period. The study spans 22 weeks approximately anddocuments the safety and efficacy of the addition of HDV to meal-timelispro, and the improved dosing of basal degludec insulin to minimizethe incidence and severity of hypoglycemia and improving HbA1c levels.

During the study, the subjects are monitored for blood glucose level,including signs of hypoglycemia. The amounts of bolus and basal insulinsprovided to the subjects are then titrated so as to ensure god glycemiccontrol without occurrence of significant hypoglycemia. This may involvereduction or increase in doses of basal insulin administered to thesubject, depending on the measured biological markers.

Example 3: Hepatic Insulin Delivery to Minimize Hypoglycemic Events inPersons with Type-1 Diabetes

Subcutaneous (SC) insulin is non-physiologic, since pancreatic insulingoes first to the liver. The present study was designed to determinewhether delivery of Hepatic Directed Vesicles (HDV) admixed withlispro/HUMALOG® (HDV-L) decreases hypoglycemia in well controlledpatients on multiple daily injections (MDI) with type 1 diabetes (T1D)using unblinded Dexcom G6 continuous glucose monitoring (CGM).

This study was a 6-month (mo) open label study of prandial insulin(lispro, 3 mo, then HDV-L, 3 mo) with basal insulin degludec (TRESIBA®)and unblinded continuous glucose monitoring (CGM) in T1D with baselineA1C 6.5-8.5%. Insulin dosing, hypoglycemia, and daily glucose controlwere among the monitored parameters.

In this study the target fasting blood glucose was 80-100 mg/dL. At 3 mosubjects were randomized to −10% or −40% basal dose to encouragetitration with HDV-L. Physicians titrated basal insulin weekly. Ahypoglycemic event was defined as ≥15 min of CGM ≤54 mg/dL.

Insulin Dosing:

At study end, degludec dosage was similar, while HDV-L dose increased0.03 U/kg/day (+13%, p=0.023) compared to optimal lispro.

There was no change in basal insulin between optimal standard of careand optimal HDV treatment, however there were significant increases inbolus insulin dosing between optimal standard of care and optimal HDVtreatment: for the −10% group: +0.02 U/kg/day; for the −40% group: +0.06U/kg/day. Basal insulin ratio was inverted in the −40% treatment groupto more bolus than basal insulin.

A1c:

In 61 enrollees, the mean baseline A1C (%) was 7.3. A1C was 6.9 after 3mo lispro optimization, and 7.0 after 3 mo HDV-L optimization. Nosignificant change in A1C between optimal standard of care and optimalHDV treatment was thus observed.

Mean Daily Glucose:

No change in mean daily glucose (<5 mg/dL) over 24 hr day, at night orduring the day.

Hypoglycemic Events:

At baseline there were 1.11 hypoglycemic events per week (EPW) (1.04Daytime “DT” and 1.39 Nighttime “NT” EPW), which decreased by 11% to0.99 EPW (0.93 DT and 1.10 NT EPW) at 3 mo. At end of study, the switchto HDV-L resulted in a further 20% decrease in events to 0.80 EPW(p=0.18; 0.86 DT, and 0.75 NT EPW p=0.08).

Both −10% and −40% treatment groups demonstrated a decrease inhypoglycemic events per week. −40% group consistently had greaterbenefit −24 hour: −26% vs. −13%; Night Time: −42% vs. −21%; Day Time:−17% vs. +1%.

Weight:

Weight (−40% group lost 0.5 kg at the end of the study)

The switch to HDV-L from lispro reduced hypoglycemia numerically,especially nocturnally, without a significant further change in A1C.This further hypoglycemia reduction is consistent with the putativebenefit of targeting insulin to the liver by inducing glycogen storagepostprandially, which may lead to decrease in hypoglycemia especially atnight. In certain embodiments, by changing the bolus to basal insulinratio (such as, by decreasing the basal dose and increasing the bolusdose), the patient can achieve an overall reduction in hypoglycemiaevents. In other embodiments, by changing the bolus to basal insulinratio (such as, by decreasing the basal dose and increasing the bolusdose), the patient can simultaneously reduce HbA1C, total cholesterol,weight, and incidence of serious hypoglycemia events. It is thusconcluded that hepatic-directed insulin delivery in persons with T1Dhelps to restore hepatic physiology.

Enumerated Embodiments:

The following exemplary embodiments are provided, the numbering of whichis not to be construed as designating levels of importance.

Embodiment 1 provides a method of optimizing the amount of bolus insulinand basal insulin to be administered to a subject having diabetesmellitus and/or a metabolic derangement, wherein the subject isadministered an amount of a bolus insulin HDV composition comprising alipid-based nanoparticle, wherein the bolus insulin is dispersed withinthe nanoparticle, wherein the subject is further administered an amountof basal insulin, the method comprising varying the administered amountof the bolus insulin HDV composition and the administered amount of thebasal insulin so as to identify the optimized amount of the bolusinsulin HDV composition and the optimized amount of the basal insulin tobe administered to the subject to afford therapeutically effective bloodglucose control without significant hypoglycemia; wherein thenanoparticle is enclosed by a bipolar lipid membrane comprisingcholesterol, dicetyl phosphate, an amphipathic lipid, and a hepatocytereceptor binding molecule; wherein the amphipathic lipid comprises atleast one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at least onehepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.

Embodiment 2 provides the method of Embodiment 1, wherein the optimizedamount of basal insulin to be administered to the subject to affordtherapeutically effective blood glucose control without significanthypoglycemia is lower when the subject is administered the bolus insulinHDV composition as compared to when the subject is administered bolusinsulin which is not part of a HDV composition.

Embodiment 3 provides the method of any of Embodiments 1-2, wherein theoptimized amount of bolus insulin to be administered to the subject soas to afford therapeutically effective blood glucose control withoutsignificant hypoglycemia is lower when the subject is administered thebolus insulin HDV composition as compared to when the subject isadministered bolus insulin which is not part of a HDV composition.

Embodiment 4 provides the method of any of Embodiments 1-3, wherein theinsulin ratio between the optimized administered bolus insulin HDVcomposition and the optimized administered basal insulin is a functionof the subject's HbA1c level.

Embodiment 5 provides the method of any of Embodiments 1-4, wherein theinsulin ratio between the optimized administered bolus insulin HDVcomposition and the optimized administered basal insulin is equal to orlower than 1:1 when the subject has >8.5% HbA1c.

Embodiment 6 provides the method of any of Embodiments 1-4, wherein theinsulin ratio between the optimized administered bolus insulin HDVcomposition and the optimized administered basal insulin is equal to orhigher than 1:1 when the subject has <8.5% HbA1c.

Embodiment 7 provides the method of any of Embodiments 1-4, wherein theinsulin ratio between the optimized administered bolus insulin HDVcomposition and the optimized administered basal insulin ranges fromabout 1:0.6 to about 1:0.9 when the subject has <8.5% HbA1c.

Embodiment 8 provides a method of optimizing the amount of bolus insulinand basal insulin to be administered to a subject having diabetesmellitus and/or a metabolic derangement, wherein the subject isoriginally administered an amount of bolus insulin and an amount ofbasal insulin such that the diabetes is well controlled in the subject,the method comprising reducing the amount of basal insulin administeredto the subject and varying the administered amount of a bolus insulinHDV composition so as to identify the optimized amount of the bolusinsulin HDV composition and the optimized amount of the basal insulin tobe administered to the subject such that the diabetes is well controlledin the subject; wherein the bolus insulin HDV composition comprises alipid-based nanoparticle, wherein the bolus insulin is dispersed withinthe nanoparticle, wherein the nanoparticle is enclosed by a bipolarlipid membrane comprising cholesterol, dicetyl phosphate, an amphipathiclipid, and a hepatocyte receptor binding molecule; wherein theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-di stearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphate,and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at leastone hepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.

Embodiment 9 provides the method of Embodiment 8, wherein the subjecthas about 6.5-8.5% A1C.

Embodiment 10 provides the method of any of Embodiments 8-9, wherein thesubject has 80-100 mg/dL fasting blood sugar.

Embodiment 11 provides the method of any of Embodiments 8-10, whereinthe subject experiences fewer hypoglycemia as compared to the treatmentwithout HDV.

Embodiment 12 provides the method of any of Embodiments 8-11, whereinthe reduction in the amount of bolus insulin ranges from about 1% toabout 80%.

Embodiment 13 provides the method of any of Embodiments 8-12, whereinthe reduction in the amount of bolus insulin ranges from about 10% toabout 40%.

Embodiment 14 provides the method of any of Embodiments 8-13, whereinthe subject experiences weight loss as compared to the treatment withoutHDV.

Embodiment 15 provides the method of any of Embodiments 8-14, whereinthe subject does not experience significant iatrogenic hyperinsulinemia.

Embodiment 16 provides the method of any of Embodiments 8-15, whereinthe basal insulin HDV composition further comprises a GLP-1 agonistand/or serotonin.

Embodiment 17 provides the method of any of Embodiments 8-16, whereinthe GLP-1 agonist comprises liraglutide, semaglutide, or repaglinide.

Embodiment 18 provides the method of any of Embodiments 8-17, whereinthe basal insulin is formulated in a composition comprising alipid-based nanoparticle, wherein the basal insulin is dispersed withinthe nanoparticle; wherein the nanoparticle is enclosed by a bipolarlipid membrane comprising cholesterol, dicetyl phosphate, an amphipathiclipid, and a hepatocyte receptor binding molecule; wherein theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at least onehepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.

Embodiment 19 provides the method of any of Embodiments 1-18, whereinthe basal insulin is administered continuously to the subject over aperiod of at least 24 hours.

Embodiment 20 provides the method of any of Embodiments 1-19, whereinthe composition is administered continuously to the subject using apump.

Embodiment 21 provides the method of any of Embodiments 1-20, whereinthe subject has a hemoglobin A1c level equal to or lower than 8.5%.

Embodiment 22 provides the method of any of Embodiments 1-21, whereinthe subject has a hemoglobin A1c level equal to or lower than about8.5%, and equal to or greater than 6.5%.

Embodiment 23 provides the method of any of Embodiments 1-22, whereinthe membrane further comprises at least one agent selected from thegroup consisting of a stabilizer and stearoyl lysophosphatidylcholine.

Embodiment 24 provides the method of any of Embodiments 1-23, whereinthe stabilizer is selected from the group consisting of m-cresol, benzylalcohol, methyl 4-hydroxybenzoate, thiomersal, and butylatedhydroxytoluene (2,6-di-tert-butyl-4-methylphenol).

Embodiment 25 provides the method of any of Embodiments 23-24, whereinthe stabilizer ranges from about 10% to about 25% (w/w) in the membrane.

Embodiment 26 provides the method of Embodiment 23, wherein the stearoyllysophosphatidylcholine ranges from about 5% to about 30% (w/w) in themembrane.

Embodiment 27 provides the method of any of Embodiments 1-26, whereinthe insulin is covalently bound to the nanoparticle.

Embodiment 28 provides the method of any of Embodiments 1-26, whereinthe insulin is not covalently bound to the nanoparticle.

Embodiment 29 provides the method of any of Embodiments 1-28 wherein theinsulin is suspended in an aqueous solution comprising a free dissolvedinsulin that is not dispersed within the nanoparticle.

Embodiment 30 provides the method of Embodiment 29, wherein thenanoparticle-dispersed insulin and the free dissolved insulin areindependently selected from the group consisting of insulin lispro,insulin aspart, regular insulin, insulin glargine, insulin zinc,extended human insulin zinc suspension, isophane insulin, human bufferedregular insulin, insulin glulisine, recombinant human regular insulin,recombinant human insulin isophane, insulin detemir, biphasic humaninsulin, and insulin deglude, and any combinations thereof.

Embodiment 31 provides the method of any of Embodiments 1-30, whereinthe amphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

Embodiment 32 provides the method of any of Embodiments 1-31, whereinthe hepatocyte receptor binding molecule comprises biotin.

Embodiment 33 provides the method of Embodiment 32, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of N-hydroxysuccinimide(NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoylbiocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE(2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.

Embodiment 34 provides the method of Embodiment 33, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE.

Embodiment 35 provides the method of any of Embodiments 1-34, whereinthe composition further comprises cellulose acetate phthalate, which isat least partially bound to the therapeutic agent dispersed within thenanoparticle.

Embodiment 36 provides the method of any of Embodiments 1-35, whereinthe composition further comprises at least one charged organic moleculebound to the therapeutic agent dispersed within the nanoparticle,wherein the charged organic molecule is at least one selected from thegroup consisting of protamines, polylysine, poly (arg-pro-thr)n in amole ratio of 1:1:1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6:1,histones, sugar polymers comprising a primary amino group,polynucleotides with primary amino groups, proteins comprising aminoacid residues with carboxyl (COO—) or sulfhydral (S—) functional groups,and acidic polymers.

Embodiment 37 provides the method of any of Embodiments 1-36, whereinthe cholesterol ranges from about 5% to about 25% (w/w) in the membrane.

Embodiment 38 provides the method of any of Embodiments 1-37, whereinthe dicetyl phosphate ranges from about 10% to about 25% (w/w) in themembrane.

Embodiment 39 provides the method of any of Embodiments 1-38, whereinthe DSPC ranges from about 40% to about 75% (w/w) in the membrane.

Embodiment 40 provides the method of any of Embodiments 1-39, whereinthe hepatocyte receptor binding molecule ranges from about 0.5% to about10% (w/w) in the membrane.

Embodiment 41 provides the method of Embodiment 23, wherein the amountof the stearoyl lysophosphatidylcholine in the membrane is about 5%-30%(w/w) of the amount of DSPC in the membrane.

Embodiment 42 provides the method of Embodiment 23, wherein the membranecomprises one of the following: (a) cholesterol, dicetyl phosphate,DSPC, stearoyl lysophosphatidylcholine, m-cresol, and at least oneselected from the group consisting of biotin DHPE and biotin-X-DHPE; (b)cholesterol, dicetyl phosphate, DSPC, m-cresol, and at least oneselected from the group consisting of biotin DHPE and biotin-X-DHPE; and(c) cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE.

Embodiment 43 provides the method of Embodiment 23, wherein the membranecomprises cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w/w) ratioselected from the group consisting of: (a) about9.4:18.1:56.8:14.1:0.0:1.5; (b) about 7.7:15.0:58.6:0.0:17.4:1.3; and(c) about 8.4:16.2:47.5:7.6:19.0:1.3.

Embodiment 44 provides the method of any of Embodiments 1-43, whereinthe subject has Type 1 diabetes, Type 2 diabetes, and/or a metabolicderangement.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A method of eliminating or minimizing events of iatrogenichyperinsulinemia or hypoglycemia in a subject having diabetes mellitusor a metabolic derangement, wherein the subject is originallyadministered an amount of bolus non-HDV insulin and an amount of basalinsulin such that the subject originally has greater than about 8.5%HbA1c, the method comprising: administering to the subject a bolusinsulin HDV composition in place of the original bolus non-HDV insulin,wherein the amount of insulin in the bolus insulin HDV composition islower than in the original bolus non-HDV insulin; administering to thesubject a basal insulin; varying the administered amount of the bolusinsulin HDV composition and the administered amount of the basal insulinso as to identify the optimized amount of the bolus insulin HDVcomposition and the optimized amount of the basal insulin to beadministered to the subject to afford therapeutically effective bloodglucose control without, or with minimized, events of iatrogenichyperinsulinemia or hypoglycemia; wherein the bolus insulin HDVcomposition comprises a lipid-based nanoparticle, wherein the bolusinsulin is dispersed within the nanoparticle, wherein the nanoparticleis enclosed by a bipolar lipid membrane comprising cholesterol, dicetylphosphate, an amphipathic lipid, and a hepatocyte receptor bindingmolecule; wherein the amphipathic lipid comprises at least one selectedfrom the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphate,and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at leastone hepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.
 2. (canceled)
 3. The method of claim 1, whereinthe insulin ratio between the optimized administered bolus insulin HDVcomposition and the optimized administered basal insulin is equal to orlower than 1:1. 4-5. (canceled)
 6. A method of eliminating or minimizingevents of iatrogenic hyperinsulinemia or hypoglycemia in a subjecthaving diabetes mellitus or a metabolic derangement, wherein the subjectis originally administered an amount of bolus non-HDV insulin and anamount of basal insulin such that the subject originally has about6.5-8.5% HbA1c, the method comprising: administering to the subject abolus insulin HDV composition in place of the original bolus non-HDVinsulin; administering to the subject a reducing amount of basal insulinas compared to the amount of basal insulin originally administered tothe subject; and varying the administered amount of the bolus insulinHDV composition so as to identify the optimized amount of the bolusinsulin HDV composition and the optimized amount of the basal insulin tobe administered to the subject such that the diabetes is well controlledin the subject without, or with minimized, events of iatrogenichyperinsulinemia or hypoglycemia; wherein the bolus insulin HDVcomposition comprises a lipid-based nanoparticle, wherein the bolusinsulin is dispersed within the nanoparticle, wherein the nanoparticleis enclosed by a bipolar lipid membrane comprising cholesterol, dicetylphosphate, an amphipathic lipid, and a hepatocyte receptor bindingmolecule; wherein the amphipathic lipid comprises at least one selectedfrom the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at least onehepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.
 7. The method of claim 6, wherein the subjectbefore optimization has about 80-100 mg/dL fasting blood sugar.
 8. Themethod of claim 6, wherein at least one of the following results isobserved upon optimization: (a) the subject experiences fewerhypoglycemia as compared to the treatment without HDV; (b) the subjectexperiences weight loss as compared to the treatment without HDV 9.(canceled)
 10. The method of claim 6, wherein the bolus insulin HDVcomposition further comprises a GLP-1 agonist or serotonin.
 11. Themethod of claim 10, wherein the GLP-1 agonist comprises liraglutide,semaglutide, or repaglinide.
 12. The method of claim 6, wherein thebasal insulin is formulated in a composition comprising a lipid-basednanoparticle, wherein the basal insulin is dispersed within thenanoparticle; wherein the nanoparticle is enclosed by a bipolar lipidmembrane comprising cholesterol, dicetyl phosphate, an amphipathiclipid, and a hepatocyte receptor binding molecule; wherein theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the at least onehepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.
 13. The method of claim 6, wherein the basalinsulin is administered continuously to the subject over a period of atleast 24 hours.
 14. The method of claim 6, wherein the composition isadministered continuously to the subject using a pump.
 15. (canceled)16. The method of claim 6, wherein the membrane further comprises atleast one agent selected from the group consisting of a stabilizer andstearoyl lysophosphatidylcholine.
 17. The method of claim 16, whereinthe stabilizer is selected from the group consisting of m-cresol, benzylalcohol, methyl 4-hydroxybenzoate, thiomersal, and butylatedhydroxytoluene (2,6-di-tert-butyl-4-methylphenol).
 18. The method ofclaim 16, wherein the stabilizer ranges from about 10% to about 25%(w/w) in the membrane, or the stearoyl lysophosphatidylcholine rangesfrom about 5% to about 30% (w/w) in the membrane.
 19. The method ofclaim 6, wherein the insulin is covalently bound to the nanoparticle orthe insulin is not covalently bound to the nanoparticle.
 20. The methodof claim 6, wherein the nanoparticle is suspended in an aqueous solutioncomprising a free dissolved insulin that is not dispersed within thenanoparticle.
 21. The method of claim 20, wherein thenanoparticle-dispersed insulin and the free dissolved insulin areindependently selected from the group consisting of insulin lispro,insulin aspart, regular insulin, insulin glargine, insulin zinc,extended human insulin zinc suspension, isophane insulin, human bufferedregular insulin, insulin glulisine, recombinant human regular insulin,recombinant human insulin isophane, insulin detemir, biphasic humaninsulin, and insulin deglude, and any combinations thereof.
 22. Themethod of claim 6, wherein the amphipathic lipid comprises at least oneselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).
 23. Themethod of claim 6, wherein the hepatocyte receptor binding moleculecomprises biotin.
 24. The method of claim 23, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of N-hydroxysuccinimide(NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoylbiocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE(2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.
 25. The method of claim 23, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE.
 26. The method of claim 6, wherein at least one applies:(a) the cholesterol ranges from about 5% to about 25% (w/w) in themembrane; (b) the dicetyl phosphate ranges from about 10% to about 25%(w/w) in the membrane; (c) the DSPC ranges from about 40% to about 75%(w/w) in the membrane; (d) the hepatocyte receptor binding moleculeranges from about 0.5% to about 10% (w/w) in the membrane.
 27. Themethod of claim 16, wherein the amount of the stearoyllysophosphatidylcholine in the membrane is about 5%-30% (w/w) of theamount of DSPC in the membrane.
 28. The method of claim 16, wherein themembrane comprises one of the following: (a) cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; (b) cholesterol, dicetyl phosphate, DSPC, m-cresol, andat least one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; and (c) cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE.
 29. The method of claim 16,wherein the membrane comprises cholesterol, dicetyl phosphate, DSPC,stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w/w)ratio selected from the group consisting of: (a) about9.4:18.1:56.8:14.1:0.0:1.5; (b) about 7.7:15.0:58.6:0.0:17.4:1.3; and(c) about 8.4:16.2:47.5:7.6:19.0:1.3.
 30. (canceled)
 31. The method ofclaim 1, wherein the bolus insulin HDV composition further comprises aGLP-1 agonist or serotonin.
 32. The method of claim 31, wherein theGLP-1 agonist comprises liraglutide, semaglutide, or repaglinide. 33.The method of claim 1, wherein the basal insulin is administeredcontinuously to the subject over a period of at least 24 hours.
 34. Themethod of claim 1, wherein the composition is administered continuouslyto the subject using a pump.
 35. The method of claim 1, wherein themembrane further comprises at least one agent selected from the groupconsisting of a stabilizer and stearoyl lysophosphatidylcholine.
 36. Themethod of claim 35, wherein the stabilizer is selected from the groupconsisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate,thiomersal, and butylated hydroxytoluene(2,6-di-tert-butyl-4-methylphenol).
 37. The method of claim 35, whereinthe stabilizer ranges from about 10% to about 25% (w/w) in the membrane,or the stearoyl lysophosphatidylcholine ranges from about 5% to about30% (w/w) in the membrane.
 38. The method of claim 1, wherein theinsulin is covalently bound to the nanoparticle or the insulin is notcovalently bound to the nanoparticle.
 39. The method of claim 1, whereinthe nanoparticle is suspended in an aqueous solution comprising a freedissolved insulin that is not dispersed within the nanoparticle.
 40. Themethod of claim 39, wherein the nanoparticle-dispersed insulin and thefree dissolved insulin are independently selected from the groupconsisting of insulin lispro, insulin aspart, regular insulin, insulinglargine, insulin zinc, extended human insulin zinc suspension, isophaneinsulin, human buffered regular insulin, insulin glulisine, recombinanthuman regular insulin, recombinant human insulin isophane, insulindetemir, biphasic human insulin, and insulin deglude, and anycombinations thereof.
 41. The method of claim 1, wherein the amphipathiclipid comprises at least one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).
 42. Themethod of claim 1, wherein the hepatocyte receptor binding moleculecomprises biotin.
 43. The method of claim 42, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of N-hydroxysuccinimide(NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoylbiocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE(2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.
 44. The method of claim 42, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE.
 45. The method of claim 1, wherein at least one applies:(a) the cholesterol ranges from about 5% to about 25% (w/w) in themembrane; (b) the dicetyl phosphate ranges from about 10% to about 25%(w/w) in the membrane; (c) the DSPC ranges from about 40% to about 75%(w/w) in the membrane; (d) the hepatocyte receptor binding moleculeranges from about 0.5% to about 10% (w/w) in the membrane.
 46. Themethod of claim 35, wherein the amount of the stearoyllysophosphatidylcholine in the membrane is about 5%-30% (w/w) of theamount of DSPC in the membrane.
 47. The method of claim 35, wherein themembrane comprises one of the following: (a) cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; (b) cholesterol, dicetyl phosphate, DSPC, m-cresol, andat least one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; and (c) cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE.
 48. The method of claim 35,wherein the membrane comprises cholesterol, dicetyl phosphate, DSPC,stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w/w)ratio selected from the group consisting of: (a) about9.4:18.1:56.8:14.1:0.0:1.5; (b) about 7.7:15.0:58.6:0.0:17.4:1.3; and(c) about 8.4:16.2:47.5:7.6:19.0:1.3.